rabbit anti n terminal nebulin antibody  (Millipore)


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    Structured Review

    Millipore rabbit anti n terminal nebulin antibody
    Knockdown of <t>nebulin</t> in primary cultures of chick skeletal myotubes results in a loss of CapZ at the Z-disc. (A) Myotubes were triple stained with antibodies to N-terminal nebulin, CapZ, and α-actinin 3 d after siRNA treatment. Treatment with nebulin-specific siRNA resulted in a dramatic decrease in nebulin staining and a reduction in the amount of CapZ at the Z-disc, with α-actinin only partially perturbed. (B) Knockdown of nebulin also resulted in phalloidin staining along the entire thin filament, not just at the Z-disc (arrowheads) and pointed ends (arrows) as observed in control cells. Myotubes were costained with an anti-C-terminal nebulin antibody. Bar, 10 μm. (C) RT-PCR consistently showed a ≥70% reduction in nebulin transcript levels in chick skeletal myotubes 1 d after treatment with nebulin-specific (KD) versus control (C) siRNA, whereas GAPDH transcript levels were similar in both samples. (D) Western blot analysis revealed a ≥90% decrease in nebulin protein levels (arrow) in chick skeletal myotubes two days after treatment with nebulin-specific (KD) versus control (C) siRNA by using an <t>anti-N-terminal</t> nebulin antibody.
    Rabbit Anti N Terminal Nebulin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 19407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc"

    Article Title: Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-07-0690

    Knockdown of nebulin in primary cultures of chick skeletal myotubes results in a loss of CapZ at the Z-disc. (A) Myotubes were triple stained with antibodies to N-terminal nebulin, CapZ, and α-actinin 3 d after siRNA treatment. Treatment with nebulin-specific siRNA resulted in a dramatic decrease in nebulin staining and a reduction in the amount of CapZ at the Z-disc, with α-actinin only partially perturbed. (B) Knockdown of nebulin also resulted in phalloidin staining along the entire thin filament, not just at the Z-disc (arrowheads) and pointed ends (arrows) as observed in control cells. Myotubes were costained with an anti-C-terminal nebulin antibody. Bar, 10 μm. (C) RT-PCR consistently showed a ≥70% reduction in nebulin transcript levels in chick skeletal myotubes 1 d after treatment with nebulin-specific (KD) versus control (C) siRNA, whereas GAPDH transcript levels were similar in both samples. (D) Western blot analysis revealed a ≥90% decrease in nebulin protein levels (arrow) in chick skeletal myotubes two days after treatment with nebulin-specific (KD) versus control (C) siRNA by using an anti-N-terminal nebulin antibody.
    Figure Legend Snippet: Knockdown of nebulin in primary cultures of chick skeletal myotubes results in a loss of CapZ at the Z-disc. (A) Myotubes were triple stained with antibodies to N-terminal nebulin, CapZ, and α-actinin 3 d after siRNA treatment. Treatment with nebulin-specific siRNA resulted in a dramatic decrease in nebulin staining and a reduction in the amount of CapZ at the Z-disc, with α-actinin only partially perturbed. (B) Knockdown of nebulin also resulted in phalloidin staining along the entire thin filament, not just at the Z-disc (arrowheads) and pointed ends (arrows) as observed in control cells. Myotubes were costained with an anti-C-terminal nebulin antibody. Bar, 10 μm. (C) RT-PCR consistently showed a ≥70% reduction in nebulin transcript levels in chick skeletal myotubes 1 d after treatment with nebulin-specific (KD) versus control (C) siRNA, whereas GAPDH transcript levels were similar in both samples. (D) Western blot analysis revealed a ≥90% decrease in nebulin protein levels (arrow) in chick skeletal myotubes two days after treatment with nebulin-specific (KD) versus control (C) siRNA by using an anti-N-terminal nebulin antibody.

    Techniques Used: Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

    2) Product Images from "Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket"

    Article Title: Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket

    Journal: eLife

    doi: 10.7554/eLife.18492

    Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004
    Figure Legend Snippet: Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004

    Techniques Used: Activation Assay, Staining, Western Blot, Mouse Assay, Expressing

    3) Product Images from "Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death"

    Article Title: Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.412

    AUR/BSO or AUR/ERA cotreatment triggers BAX/BAK activation and loss of MMP. ( a ) RMS cells were treated for 15 h (RH30) and 18 h (RD) with 1 μ M AUR and/or 1 μ M BSO and/or ERA (RH30: 1 μ M, RD: 2 μ M). BAX/BAK activation was assessed by immunoprecipitation using a conformation-specific anti-BAX or anti-BAK antibody and expression of BAX and BAK was analyzed by Western blotting, GAPDH and β -actin served as loading control. ( b ) RMS cells were transiently transfected with siRNA against BAX and BAK or non-targeting control siRNA. Protein expression of BAX and BAK was assessed by Western blotting 48 h after knockdown. β-actin served as loading control. ( c ) RMS cells were transiently transfected with siRNA against BAX and BAK or non-targeting control siRNA and were treated for 24 h (RH30) and 48 h (RD) after knockdown with 1 μ M AUR and 1 μ M BSO or ERA (RH30: 1 μ M, RD: 2 μ M). Cell death was determined by PI staining using flow cytometry. Mean and S.D. of at least three independent experiments carried out in triplicate are shown; * P ≤0.05, ** P ≤0.01. ( d ) RMS cells were treated for 15 h (RH30) and 18 h (RD) with 1 μ M AUR and/or 1 μ M BSO and/or ERA (RH30: 1 μ M, RD: 2 μ M). Loss of MMP was determined by FACS analysis of the viable cell population using the fluorescent dye TMRM. Mean and S.D. of at least three independent experiments carried out in triplicate are shown; * P ≤0.05, ** P ≤0.01
    Figure Legend Snippet: AUR/BSO or AUR/ERA cotreatment triggers BAX/BAK activation and loss of MMP. ( a ) RMS cells were treated for 15 h (RH30) and 18 h (RD) with 1 μ M AUR and/or 1 μ M BSO and/or ERA (RH30: 1 μ M, RD: 2 μ M). BAX/BAK activation was assessed by immunoprecipitation using a conformation-specific anti-BAX or anti-BAK antibody and expression of BAX and BAK was analyzed by Western blotting, GAPDH and β -actin served as loading control. ( b ) RMS cells were transiently transfected with siRNA against BAX and BAK or non-targeting control siRNA. Protein expression of BAX and BAK was assessed by Western blotting 48 h after knockdown. β-actin served as loading control. ( c ) RMS cells were transiently transfected with siRNA against BAX and BAK or non-targeting control siRNA and were treated for 24 h (RH30) and 48 h (RD) after knockdown with 1 μ M AUR and 1 μ M BSO or ERA (RH30: 1 μ M, RD: 2 μ M). Cell death was determined by PI staining using flow cytometry. Mean and S.D. of at least three independent experiments carried out in triplicate are shown; * P ≤0.05, ** P ≤0.01. ( d ) RMS cells were treated for 15 h (RH30) and 18 h (RD) with 1 μ M AUR and/or 1 μ M BSO and/or ERA (RH30: 1 μ M, RD: 2 μ M). Loss of MMP was determined by FACS analysis of the viable cell population using the fluorescent dye TMRM. Mean and S.D. of at least three independent experiments carried out in triplicate are shown; * P ≤0.05, ** P ≤0.01

    Techniques Used: Activation Assay, Immunoprecipitation, Expressing, Western Blot, Transfection, Staining, Flow Cytometry, Cytometry, FACS

    Scheme of AUR/BSO- or AUR/ERA-triggered synergistic cell death. AUR inhibits TrxR, which leads to upregulation of GSH, inhibiting AUR. ERA or BSO cause depletion of GSH levels, thereby counteracting the AUR-mediated upregulation of GSH and increasing AUR's cytotoxic activity. This leads to proteasome inhibition and subsequently to accumulation of ubiquitinated NOXA and MCL-1, followed by activation of BAX/BAK and caspases. See text for more details
    Figure Legend Snippet: Scheme of AUR/BSO- or AUR/ERA-triggered synergistic cell death. AUR inhibits TrxR, which leads to upregulation of GSH, inhibiting AUR. ERA or BSO cause depletion of GSH levels, thereby counteracting the AUR-mediated upregulation of GSH and increasing AUR's cytotoxic activity. This leads to proteasome inhibition and subsequently to accumulation of ubiquitinated NOXA and MCL-1, followed by activation of BAX/BAK and caspases. See text for more details

    Techniques Used: Activity Assay, Inhibition, Activation Assay

    4) Product Images from "Deubiquitinase inhibitor PR-619 reduces Smad4 expression and suppresses renal fibrosis in mice with unilateral ureteral obstruction"

    Article Title: Deubiquitinase inhibitor PR-619 reduces Smad4 expression and suppresses renal fibrosis in mice with unilateral ureteral obstruction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202409

    PR-619 suppresses UUO-induced ECM deposition. The same samples in Fig 2 were used to examine collagen 1, collagen 3, and fibronectin as ECM proteins. (A) Collagen 1, collagen 3, and fibronectin mRNA levels were determined by qRT-PCR in mice with UUO with or without PR-619 administration. GAPDH was used as an internal control. (B) Typical western blot analysis demonstrating the level of fibronectin protein expression. The graph shows the expression level quantified by densitometry and normalized to GAPDH. (C) Representative images showing immunostaining for collagen 1, collagen 3, and fibronectin. Quantification is shown in the right panel. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. * P
    Figure Legend Snippet: PR-619 suppresses UUO-induced ECM deposition. The same samples in Fig 2 were used to examine collagen 1, collagen 3, and fibronectin as ECM proteins. (A) Collagen 1, collagen 3, and fibronectin mRNA levels were determined by qRT-PCR in mice with UUO with or without PR-619 administration. GAPDH was used as an internal control. (B) Typical western blot analysis demonstrating the level of fibronectin protein expression. The graph shows the expression level quantified by densitometry and normalized to GAPDH. (C) Representative images showing immunostaining for collagen 1, collagen 3, and fibronectin. Quantification is shown in the right panel. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. * P

    Techniques Used: Quantitative RT-PCR, Mouse Assay, Western Blot, Expressing, Immunostaining

    5) Product Images from "Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina"

    Article Title: Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2618-05.2006

    Decreased levels of β4 integrin in nerves of PMP22-deficient mice. A–F , Cryosections of sciatic nerves from +/+ ( A , C , E ) and PMP22−/− ( B , D , F ) mice were immunostained with monoclonal rat (rt) anti-β4 integrin ( A , B ), anti-β1 integrin ( C , D ), or polyclonal rabbit (rb) anti-laminin (Lam) ( E , F ) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface ( A , C , arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the −/− samples ( B , D , arrows). In addition, when the images were collected at the same exposure times, the level of β4-like immunoreactivity was reduced (compare A , B ). Laminin was detected at the SC basal lamina in +/+ ( E , arrow) and −/− ( F ) nerves, with thickened basal lamina (arrowheads) and a tomaculum (open arrowhead) marked in the affected sample ( F ). Nonspecific rat ( A , B , inset) and rabbit ( E , F , inset) sera serve as controls for staining specificity. Scale bar, 5 μm. G , Sciatic nerve lysates (20 μg/lane) from P10 +/+, +/−, and −/− mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin and monoclonal rat anti-β1 integrin antibodies. The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kilodaltons.
    Figure Legend Snippet: Decreased levels of β4 integrin in nerves of PMP22-deficient mice. A–F , Cryosections of sciatic nerves from +/+ ( A , C , E ) and PMP22−/− ( B , D , F ) mice were immunostained with monoclonal rat (rt) anti-β4 integrin ( A , B ), anti-β1 integrin ( C , D ), or polyclonal rabbit (rb) anti-laminin (Lam) ( E , F ) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface ( A , C , arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the −/− samples ( B , D , arrows). In addition, when the images were collected at the same exposure times, the level of β4-like immunoreactivity was reduced (compare A , B ). Laminin was detected at the SC basal lamina in +/+ ( E , arrow) and −/− ( F ) nerves, with thickened basal lamina (arrowheads) and a tomaculum (open arrowhead) marked in the affected sample ( F ). Nonspecific rat ( A , B , inset) and rabbit ( E , F , inset) sera serve as controls for staining specificity. Scale bar, 5 μm. G , Sciatic nerve lysates (20 μg/lane) from P10 +/+, +/−, and −/− mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin and monoclonal rat anti-β1 integrin antibodies. The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kilodaltons.

    Techniques Used: Mouse Assay, Laser Capture Microdissection, Staining

    6) Product Images from "Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina"

    Article Title: Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2618-05.2006

    Decreased levels of β4 integrin in nerves of PMP22-deficient mice. A–F , Cryosections of sciatic nerves from +/+ ( A , C , E ) and PMP22−/− ( B , D , F ) mice were immunostained with monoclonal rat (rt) anti-β4 integrin ( A , B ), anti-β1 integrin ( C , D ), or polyclonal rabbit (rb) anti-laminin (Lam) ( E , F ) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface ( A , C , arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the −/− samples ( B , D , arrows). In addition, when the images were collected at the same exposure times, the level of β4-like immunoreactivity was reduced (compare A , B ). Laminin was detected at the SC basal lamina in +/+ ( E , arrow) and −/− ( F ) nerves, with thickened basal lamina (arrowheads) and a tomaculum (open arrowhead) marked in the affected sample ( F ). Nonspecific rat ( A , B , inset) and rabbit ( E , F , inset) sera serve as controls for staining specificity. Scale bar, 5 μm. G , Sciatic nerve lysates (20 μg/lane) from P10 +/+, +/−, and −/− mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin and monoclonal rat anti-β1 integrin antibodies. The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kilodaltons.
    Figure Legend Snippet: Decreased levels of β4 integrin in nerves of PMP22-deficient mice. A–F , Cryosections of sciatic nerves from +/+ ( A , C , E ) and PMP22−/− ( B , D , F ) mice were immunostained with monoclonal rat (rt) anti-β4 integrin ( A , B ), anti-β1 integrin ( C , D ), or polyclonal rabbit (rb) anti-laminin (Lam) ( E , F ) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface ( A , C , arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the −/− samples ( B , D , arrows). In addition, when the images were collected at the same exposure times, the level of β4-like immunoreactivity was reduced (compare A , B ). Laminin was detected at the SC basal lamina in +/+ ( E , arrow) and −/− ( F ) nerves, with thickened basal lamina (arrowheads) and a tomaculum (open arrowhead) marked in the affected sample ( F ). Nonspecific rat ( A , B , inset) and rabbit ( E , F , inset) sera serve as controls for staining specificity. Scale bar, 5 μm. G , Sciatic nerve lysates (20 μg/lane) from P10 +/+, +/−, and −/− mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin and monoclonal rat anti-β1 integrin antibodies. The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kilodaltons.

    Techniques Used: Mouse Assay, Laser Capture Microdissection, Staining

    PMP22 is in a complex with α6β4 integrin and laminin. Sciatic nerve lysates (T lanes) from P21 +/+ mice were processed for IP, after preclearing (PC lanes) with nonspecific Igs of the appropriate isotype. Lysates were incubated with polyclonal rabbit anti-β4 integrin ( A , left, C ), anti-PMP22 ( A , right), monoclonal rat anti-α6 integrin ( B , left), or polyclonal rabbit anti-laminin ( B , right) antibodies, and captured immunoprecipitates were probed for the indicated proteins ( A–C ) as designated at the right of each blot. On reprobes (marked with asterisks), after stripping the membranes, the anti-β4 integrin and anti-laminin antibodies did not work efficiently when 1 μg/lane total (T) nerve protein was analyzed. IP with anti-β4 integrin on nerve lysates of homozygous PMP22-deficient mice is shown as a negative control ( C ). Molecular mass is in kilodaltons. PMP, PMP22; Lam, laminin; βDys, β-dystroglycan.
    Figure Legend Snippet: PMP22 is in a complex with α6β4 integrin and laminin. Sciatic nerve lysates (T lanes) from P21 +/+ mice were processed for IP, after preclearing (PC lanes) with nonspecific Igs of the appropriate isotype. Lysates were incubated with polyclonal rabbit anti-β4 integrin ( A , left, C ), anti-PMP22 ( A , right), monoclonal rat anti-α6 integrin ( B , left), or polyclonal rabbit anti-laminin ( B , right) antibodies, and captured immunoprecipitates were probed for the indicated proteins ( A–C ) as designated at the right of each blot. On reprobes (marked with asterisks), after stripping the membranes, the anti-β4 integrin and anti-laminin antibodies did not work efficiently when 1 μg/lane total (T) nerve protein was analyzed. IP with anti-β4 integrin on nerve lysates of homozygous PMP22-deficient mice is shown as a negative control ( C ). Molecular mass is in kilodaltons. PMP, PMP22; Lam, laminin; βDys, β-dystroglycan.

    Techniques Used: Mouse Assay, Incubation, Stripping Membranes, Negative Control, Laser Capture Microdissection

    7) Product Images from "SIX1 is overexpressed in endometrial carcinoma and promotes the malignant behavior of cancer cells through ERK and AKT signaling"

    Article Title: SIX1 is overexpressed in endometrial carcinoma and promotes the malignant behavior of cancer cells through ERK and AKT signaling

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.5098

    SIX1 transfection promotes proliferation and colony formation. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that SIX1 transfection upregulated cell proliferation rate in HEC1B cells, and SIX1 siRNA downregulated cell proliferation
    Figure Legend Snippet: SIX1 transfection promotes proliferation and colony formation. (A) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that SIX1 transfection upregulated cell proliferation rate in HEC1B cells, and SIX1 siRNA downregulated cell proliferation

    Techniques Used: Transfection

    Transfection efficiency of SIX1 plasmid and siRNA in HEC1B and Ishikawa endometrial carcinoma cell lines. (A) Western blot analysis and RT-qPCR showed that SIX1 mRNA expression was increased in Ishikawa cells and decreased in HEC1B cells. *P
    Figure Legend Snippet: Transfection efficiency of SIX1 plasmid and siRNA in HEC1B and Ishikawa endometrial carcinoma cell lines. (A) Western blot analysis and RT-qPCR showed that SIX1 mRNA expression was increased in Ishikawa cells and decreased in HEC1B cells. *P

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing

    Expression of SIX1 protein in endometrial carcinoma tissues. (A) Negative and (B) positive SIX1 expression in grade 1 endometrial carcinoma. (C) Negative and (D) positive expression of SIX1 in a case of grade 2 endometrial carcinoma. (E) Positive SIX1
    Figure Legend Snippet: Expression of SIX1 protein in endometrial carcinoma tissues. (A) Negative and (B) positive SIX1 expression in grade 1 endometrial carcinoma. (C) Negative and (D) positive expression of SIX1 in a case of grade 2 endometrial carcinoma. (E) Positive SIX1

    Techniques Used: Expressing

    SIX1 upregulates ERK and AKT activity. (A) SIX1 transfection was performed in HEC1B cells and siRNA knockdown was performed in Ishikawa cells. Western blot analysis showed that SIX1 transfection upregulated, while SIX1 depletion downregulated, cyclin
    Figure Legend Snippet: SIX1 upregulates ERK and AKT activity. (A) SIX1 transfection was performed in HEC1B cells and siRNA knockdown was performed in Ishikawa cells. Western blot analysis showed that SIX1 transfection upregulated, while SIX1 depletion downregulated, cyclin

    Techniques Used: Activity Assay, Transfection, Western Blot

    Expression of SIX1 in human endometrial carcinoma
    Figure Legend Snippet: Expression of SIX1 in human endometrial carcinoma

    Techniques Used: Expressing

    8) Product Images from "Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells"

    Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149728

    Double immunofluorescent staining and immunoblot analysis of the endogenous SCPX and SCP2 proteins in MA-10 cells. (A) Double immunofluorescent staining of endogenous SCPX in MA-10 cells (red), PMP70 (peroxisomal marker protein, painted in green), and VDAC1 (mitochondrial marker protein, painted in green). The nucleus was counterstained with DAPI (painted in green). Scale bar, 5 μm. (B) Immunoblot analysis of endogenous SCPX in subcellular fractions of MA-10 cell lysates. COXIV is used as a mitochondrial marker protein, and GAPDH is used as a whole cell lysate loading control. M, the protein marker, indicated in kDa; Total, whole cell lysate; Cyto, cytosolic fraction; Mito, mitochondrial-enriched fraction. (C) Double immunofluorescent staining of endogenous SCP2 in MA-10 cells (red), PMP70 (painted in green), and VDAC1 (painted in green). The nNucleus was counterstained with DAPI (painted in green). Scale bar, 5 μm. (D) Immunoblot analysis of endogenous SCP2 in subcellular fractions of MA-10 cell lysates. VDAC1 is used as a mitochondrial marker protein, and GAPDH is used as a whole cell lysate loading control. (E–F) Immunoblot analysis of the isolated MA-10 subcellular fractions identifying mitochondrial matrix protein HSP60 (E) and peroxisomal matrix protein catalase (CAT) (F). GAPDH is used as a loading control. M, the protein marker, indicated in kDa; Total, whole cell lysate; Cyto, cytosolic fractions; Mito, mitochondrial-enriched fraction.
    Figure Legend Snippet: Double immunofluorescent staining and immunoblot analysis of the endogenous SCPX and SCP2 proteins in MA-10 cells. (A) Double immunofluorescent staining of endogenous SCPX in MA-10 cells (red), PMP70 (peroxisomal marker protein, painted in green), and VDAC1 (mitochondrial marker protein, painted in green). The nucleus was counterstained with DAPI (painted in green). Scale bar, 5 μm. (B) Immunoblot analysis of endogenous SCPX in subcellular fractions of MA-10 cell lysates. COXIV is used as a mitochondrial marker protein, and GAPDH is used as a whole cell lysate loading control. M, the protein marker, indicated in kDa; Total, whole cell lysate; Cyto, cytosolic fraction; Mito, mitochondrial-enriched fraction. (C) Double immunofluorescent staining of endogenous SCP2 in MA-10 cells (red), PMP70 (painted in green), and VDAC1 (painted in green). The nNucleus was counterstained with DAPI (painted in green). Scale bar, 5 μm. (D) Immunoblot analysis of endogenous SCP2 in subcellular fractions of MA-10 cell lysates. VDAC1 is used as a mitochondrial marker protein, and GAPDH is used as a whole cell lysate loading control. (E–F) Immunoblot analysis of the isolated MA-10 subcellular fractions identifying mitochondrial matrix protein HSP60 (E) and peroxisomal matrix protein catalase (CAT) (F). GAPDH is used as a loading control. M, the protein marker, indicated in kDa; Total, whole cell lysate; Cyto, cytosolic fractions; Mito, mitochondrial-enriched fraction.

    Techniques Used: Staining, Marker, Isolation

    9) Product Images from "Vimentin Regulates Scribble Activity by Protecting It from Proteasomal Degradation"

    Article Title: Vimentin Regulates Scribble Activity by Protecting It from Proteasomal Degradation

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E08-02-0199

    Silencing of Scrib or vimentin expression in MDCK cells leads to defects in cell morphology and Golgi complex orientation during directed cell migration. (A) Aberrant morphology. Monolayers of cells treated with nontargeting (a, e, and i), vimentin (b, f, and j), Scrib (c, g, and k), or Scrib and vimentin (d, h, and l) siRNA were wounded and stained with an antibody to ZO-2 (a–d) to visualize the cell outline. Scrib (e–h) and vimentin (i–l) were stained to monitor the effectiveness of the siRNA treatment. Note how in control cells the long axis of the cells is directed toward the wound edge (bottom of the images), whereas it is random in cells treated with the specific siRNAs. (B) Monolayers of cells treated with nontargeting (a), vimentin (b), Scrib (c), or Scrib and vimentin (d) siRNA were wounded and stained with an antibody to the cis -Golgi marker GM130 (red) and DAPI (blue) to label nuclei. The wound edge is demarcated with a white line. (e) Golgi complex orientation relative to the nucleus and the migration front was quantified as described in Materials and Methods . Shown is the fraction of leading edge cellswith correctly polarized Golgi complexes that position in front of the nucleus, facing the wound. Results represent the means of three independent experiments, in which at least 400 cells where scored for each condition. Error bars, SD of the mean. A red line indicates basal levels for a random orientation of 33%. (f) Schematic representation of Golgi complex orientation. The position of Golgi complex relative to the nucleus (blue) and wound edge was determined for ∼30 individual cells for each siRNA treatment and plotted. The shaded sector from 30° to 150° faces the wound edge and is bisected perpendicular to this edge. Note how the positioning of the Golgi complex of most control siRNA-treated cells falls within this sector, whereas that of cells where vimentin, Scrib, or both had been silenced is randomized.
    Figure Legend Snippet: Silencing of Scrib or vimentin expression in MDCK cells leads to defects in cell morphology and Golgi complex orientation during directed cell migration. (A) Aberrant morphology. Monolayers of cells treated with nontargeting (a, e, and i), vimentin (b, f, and j), Scrib (c, g, and k), or Scrib and vimentin (d, h, and l) siRNA were wounded and stained with an antibody to ZO-2 (a–d) to visualize the cell outline. Scrib (e–h) and vimentin (i–l) were stained to monitor the effectiveness of the siRNA treatment. Note how in control cells the long axis of the cells is directed toward the wound edge (bottom of the images), whereas it is random in cells treated with the specific siRNAs. (B) Monolayers of cells treated with nontargeting (a), vimentin (b), Scrib (c), or Scrib and vimentin (d) siRNA were wounded and stained with an antibody to the cis -Golgi marker GM130 (red) and DAPI (blue) to label nuclei. The wound edge is demarcated with a white line. (e) Golgi complex orientation relative to the nucleus and the migration front was quantified as described in Materials and Methods . Shown is the fraction of leading edge cellswith correctly polarized Golgi complexes that position in front of the nucleus, facing the wound. Results represent the means of three independent experiments, in which at least 400 cells where scored for each condition. Error bars, SD of the mean. A red line indicates basal levels for a random orientation of 33%. (f) Schematic representation of Golgi complex orientation. The position of Golgi complex relative to the nucleus (blue) and wound edge was determined for ∼30 individual cells for each siRNA treatment and plotted. The shaded sector from 30° to 150° faces the wound edge and is bisected perpendicular to this edge. Note how the positioning of the Golgi complex of most control siRNA-treated cells falls within this sector, whereas that of cells where vimentin, Scrib, or both had been silenced is randomized.

    Techniques Used: Expressing, Migration, Staining, Marker

    Slower wound closure rates due to a less directed migration of MDCK cells treated with Scrib or vimentin siRNA. (A) Wound closure. Monolayers of cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were wounded and allowed to migrate for 16 h. Images were taken after wounding (0 h; s a–d) or 16 h of migration (e–h). The black marks at the bottom of the dishes allow alignment of the wounds. Panels shown are representative of at least three independent experiments. (B) Quantification of cell migration directionality using live cell tracking. The X-Y graphs represent migration coordinates of 10 different cells at the wound edge treated with nontargeting (a), vimentin (b), Scrib (c), or vimentin and Scrib (d) siRNA, tracked over time 4 d after siRNA transfection. Start points for the different cells were adjusted to (0,0) coordinates. Results are representative of at least three independent experiments. (e) Tortuosity was scored for at least 30 individual cells for each siRNA treatment (n = 3; p
    Figure Legend Snippet: Slower wound closure rates due to a less directed migration of MDCK cells treated with Scrib or vimentin siRNA. (A) Wound closure. Monolayers of cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were wounded and allowed to migrate for 16 h. Images were taken after wounding (0 h; s a–d) or 16 h of migration (e–h). The black marks at the bottom of the dishes allow alignment of the wounds. Panels shown are representative of at least three independent experiments. (B) Quantification of cell migration directionality using live cell tracking. The X-Y graphs represent migration coordinates of 10 different cells at the wound edge treated with nontargeting (a), vimentin (b), Scrib (c), or vimentin and Scrib (d) siRNA, tracked over time 4 d after siRNA transfection. Start points for the different cells were adjusted to (0,0) coordinates. Results are representative of at least three independent experiments. (e) Tortuosity was scored for at least 30 individual cells for each siRNA treatment (n = 3; p

    Techniques Used: Migration, Cell Tracking Assay, Transfection

    siRNA-mediated depletion of endogenous vimentin and Scrib in MDCK cells. (A) Silencing of Scrib and vimentin monitored by immunofluorescence microscopy. Scrib (a–d, red) and vimentin (e–h, white) were visualized in MDCK cells treated for 3 d with a nontargeting siRNA (a and e) or siRNAs to vimentin (b and f), Scrib (c and g), or both Scrib and vimentin (d and h). (B) Silencing of Scrib and vimentin monitored by Western blot analysis. Scrib and vimentin protein levels in lysates of cells treated with siRNA over a 6-d period were monitored by Western blot on days 2, 4, and 6. Keratin 18 was detected to monitor for equal cell lysate loading.
    Figure Legend Snippet: siRNA-mediated depletion of endogenous vimentin and Scrib in MDCK cells. (A) Silencing of Scrib and vimentin monitored by immunofluorescence microscopy. Scrib (a–d, red) and vimentin (e–h, white) were visualized in MDCK cells treated for 3 d with a nontargeting siRNA (a and e) or siRNAs to vimentin (b and f), Scrib (c and g), or both Scrib and vimentin (d and h). (B) Silencing of Scrib and vimentin monitored by Western blot analysis. Scrib and vimentin protein levels in lysates of cells treated with siRNA over a 6-d period were monitored by Western blot on days 2, 4, and 6. Keratin 18 was detected to monitor for equal cell lysate loading.

    Techniques Used: Immunofluorescence, Microscopy, Western Blot

    Proteasome-dependent degradation of Scrib is inhibited by its interaction with vimentin. (A) Vimentin expression in MDCK cells was silenced using siRNA over 3 d. Cells were subsequently reseeded to sparse and confluent cultures, and Scrib protein levels were monitored by Western blot analysis on day 4. K18 was detected to check for e qual cell lysate loading. (B) Quantitative representation of Scrib down-regulation relative to levels of vimentin silencing in MDCK cells. (C) MDCK cells expressing EGFP-hScrib WT (∼250 kDa), LRR (∼130 kDa), PDZ (∼150 kDa), or, as a negative control, EGFP alone, were treated with vimentin (+) or nontargeting (−) siRNA. hScrib expression was analyzed by Western blot using antibodies to GFP. GAPDH served as a control for equal lysate input. (D) MDCK cells expressing EGFP-hScrib WT (a–d), LRR (e–h), or PDZ (i–l) were treated with nontargeting (a, b, e, f, I, and j) or vimentin (c, d, g, h, k, and l) siRNA and EGFP-hScrib (b, f, j, d, h, and l; green) and vimentin (a, e, i, c, g, and k; red) expression was visualized by fluorescence microscopy. (E) MDCK cells exogenously expressing EGFP vimentin, ECFP-K8, EYFP-K18, or EGFP alone were analyzed by Western blot for expression of Scrib. GAPDH served as a control for equal lysate input. (F–H) Effect of a proteasome inhibitor on Scrib turnover. (F) Western blot. MDCK cells expressing EGFP-hScrib WT were treated with vimentin (+) or nontargeting (−) siRNA for 3 d and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Scrib levels and vimentin expression levels were then analyzed by Western blot. Note how in vimentin depleted cells, EGFP-hScrib (250 kDa) as well as endogenous Scrib (220 kDa) degradation is blocked by the proteasome inhibitor (also see H). Actin served as a control for equal lysate input. (G) Immunofluorescence microscopy. MDCK cells expressing EGFP-hScrib WT were treated with vimentin siRNA and subsequently, a proteasome inhibitor for 0 h (a and b) or 9 h (c and d) and EGFP-hScrib (a and c; green) and vimentin (b and d; red) expression was visualized by fluorescence microscopy. (H) Western blot for endogenous Scrib. MDCK cells were treated with vimentin (+) or nontargeting (−) siRNA and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Endogenous levels of canine Scrib and vimentin were then analyzed by Western blot. GAPDH served as a control for equal lysate input. (I) hScrib-EGFP of nontargeting or vimentin siRNA-treated MDCK cells in the 9 h presence (+) or absence (−) of proteasome inhibitor was immunoprecipitated and ubiquitinylated hScrib detected by Western blot. Normal IgG served as a negative control.
    Figure Legend Snippet: Proteasome-dependent degradation of Scrib is inhibited by its interaction with vimentin. (A) Vimentin expression in MDCK cells was silenced using siRNA over 3 d. Cells were subsequently reseeded to sparse and confluent cultures, and Scrib protein levels were monitored by Western blot analysis on day 4. K18 was detected to check for e qual cell lysate loading. (B) Quantitative representation of Scrib down-regulation relative to levels of vimentin silencing in MDCK cells. (C) MDCK cells expressing EGFP-hScrib WT (∼250 kDa), LRR (∼130 kDa), PDZ (∼150 kDa), or, as a negative control, EGFP alone, were treated with vimentin (+) or nontargeting (−) siRNA. hScrib expression was analyzed by Western blot using antibodies to GFP. GAPDH served as a control for equal lysate input. (D) MDCK cells expressing EGFP-hScrib WT (a–d), LRR (e–h), or PDZ (i–l) were treated with nontargeting (a, b, e, f, I, and j) or vimentin (c, d, g, h, k, and l) siRNA and EGFP-hScrib (b, f, j, d, h, and l; green) and vimentin (a, e, i, c, g, and k; red) expression was visualized by fluorescence microscopy. (E) MDCK cells exogenously expressing EGFP vimentin, ECFP-K8, EYFP-K18, or EGFP alone were analyzed by Western blot for expression of Scrib. GAPDH served as a control for equal lysate input. (F–H) Effect of a proteasome inhibitor on Scrib turnover. (F) Western blot. MDCK cells expressing EGFP-hScrib WT were treated with vimentin (+) or nontargeting (−) siRNA for 3 d and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Scrib levels and vimentin expression levels were then analyzed by Western blot. Note how in vimentin depleted cells, EGFP-hScrib (250 kDa) as well as endogenous Scrib (220 kDa) degradation is blocked by the proteasome inhibitor (also see H). Actin served as a control for equal lysate input. (G) Immunofluorescence microscopy. MDCK cells expressing EGFP-hScrib WT were treated with vimentin siRNA and subsequently, a proteasome inhibitor for 0 h (a and b) or 9 h (c and d) and EGFP-hScrib (a and c; green) and vimentin (b and d; red) expression was visualized by fluorescence microscopy. (H) Western blot for endogenous Scrib. MDCK cells were treated with vimentin (+) or nontargeting (−) siRNA and subsequently in the presence of a proteasome inhibitor for 0, 3, 6, or 9 h. Endogenous levels of canine Scrib and vimentin were then analyzed by Western blot. GAPDH served as a control for equal lysate input. (I) hScrib-EGFP of nontargeting or vimentin siRNA-treated MDCK cells in the 9 h presence (+) or absence (−) of proteasome inhibitor was immunoprecipitated and ubiquitinylated hScrib detected by Western blot. Normal IgG served as a negative control.

    Techniques Used: Expressing, Western Blot, Negative Control, Fluorescence, Microscopy, Immunofluorescence, Immunoprecipitation

    Silencing of Scrib and vimentin expression affects cell–cell aggregation and spreading. (A) Cell aggregation. MDCK cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were allowed to aggregate in a hanging drop and photographed (a–d). (B) Cell spreading. Cell aggregates were transferred from the hanging drop onto coverslips and allowed to adhere and spread (e–h). Note how cells treated with specific siRNAs form less compact aggregates (b–d) and show enhanced spreading (f–h) compared with control cells (a and e, respectively). Assays were carried out 4 d after siRNA transfection.
    Figure Legend Snippet: Silencing of Scrib and vimentin expression affects cell–cell aggregation and spreading. (A) Cell aggregation. MDCK cells treated with nontargeting (a and e), vimentin (b and f), Scrib (c and g), or Scrib and vimentin (d and h) siRNA were allowed to aggregate in a hanging drop and photographed (a–d). (B) Cell spreading. Cell aggregates were transferred from the hanging drop onto coverslips and allowed to adhere and spread (e–h). Note how cells treated with specific siRNAs form less compact aggregates (b–d) and show enhanced spreading (f–h) compared with control cells (a and e, respectively). Assays were carried out 4 d after siRNA transfection.

    Techniques Used: Expressing, Transfection

    10) Product Images from "RNA Interference Against Hepatic Epidermal Growth Factor Receptor Has Suppressive Effects on Liver Regeneration in Rats"

    Article Title: RNA Interference Against Hepatic Epidermal Growth Factor Receptor Has Suppressive Effects on Liver Regeneration in Rats

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.090605

    Co-immunoprecipitation analyses in treated and control rats. A: MET-EGFR co-immunoprecipitation. Cell lysates were immunoprecipitated with a mouse monoclonal MET antibody and probed with an EGFR antibody as described in Materials and Methods . An increase
    Figure Legend Snippet: Co-immunoprecipitation analyses in treated and control rats. A: MET-EGFR co-immunoprecipitation. Cell lysates were immunoprecipitated with a mouse monoclonal MET antibody and probed with an EGFR antibody as described in Materials and Methods . An increase

    Techniques Used: Immunoprecipitation

    Western blot analyses of some of the relevant targets like MET, PDGFRa, and cyclin D1 that were found to be differentially regulated by qRT-PCR analyses were validated by Western blot analyses as described in Materials and Methods . As seen in A , an up-regulation
    Figure Legend Snippet: Western blot analyses of some of the relevant targets like MET, PDGFRa, and cyclin D1 that were found to be differentially regulated by qRT-PCR analyses were validated by Western blot analyses as described in Materials and Methods . As seen in A , an up-regulation

    Techniques Used: Western Blot, Quantitative RT-PCR

    11) Product Images from "DYSLIPIDEMIA IS A MAJOR FACTOR IN STEM CELL DAMAGE INDUCED BY UNCONTROLLED LONG-TERM TYPE 2 DIABETES AND OBESITY IN THE RAT, AS SUGGESTED BY THE EFFECTS ON STEM CELL CULTURE"

    Article Title: DYSLIPIDEMIA IS A MAJOR FACTOR IN STEM CELL DAMAGE INDUCED BY UNCONTROLLED LONG-TERM TYPE 2 DIABETES AND OBESITY IN THE RAT, AS SUGGESTED BY THE EFFECTS ON STEM CELL CULTURE

    Journal: The journal of sexual medicine

    doi: 10.1016/j.jsxm.2018.09.019

    Incubation of ED-MDSC with increasing aged OZ serum caused a concentration dependent expression of myostatin protein, a pro-lipofibrotic and muscle mass inhibitor, but the incubation with increasing glucose exerted an opposite effect.
    Figure Legend Snippet: Incubation of ED-MDSC with increasing aged OZ serum caused a concentration dependent expression of myostatin protein, a pro-lipofibrotic and muscle mass inhibitor, but the incubation with increasing glucose exerted an opposite effect.

    Techniques Used: Incubation, Concentration Assay, Expressing

    12) Product Images from "Mena binds ?5 integrin directly and modulates ?5?1 function"

    Article Title: Mena binds ?5 integrin directly and modulates ?5?1 function

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201202079

    Expression and distribution of Mena and α5 in cells lacking either protein.  (A) Western blots of lysates from subdermal fibroblasts isolated from Mena FLOXED  (Mena F/F , homozygous for VASP deletion) or α5 FLOXED (α5 F/F ) mice, 48 h after infection with GFP or GFP-Cre adenovirus, and probed with anti-α5, -Mena, -VASP, or -tubulin. (B) Quantitative PCR analysis of Mena mRNA levels in α5 FLOXED  and α5-null fibroblasts. Immunofluorescence of Mena F/F  (C) or α5 F/F (D) cells after infection with GFP or GFP-Cre adenovirus. Panels on the right show enlarged views of the boxed regions. Error bars indicate mean ± SEM. Bar, 10 µm.
    Figure Legend Snippet: Expression and distribution of Mena and α5 in cells lacking either protein. (A) Western blots of lysates from subdermal fibroblasts isolated from Mena FLOXED (Mena F/F , homozygous for VASP deletion) or α5 FLOXED (α5 F/F ) mice, 48 h after infection with GFP or GFP-Cre adenovirus, and probed with anti-α5, -Mena, -VASP, or -tubulin. (B) Quantitative PCR analysis of Mena mRNA levels in α5 FLOXED and α5-null fibroblasts. Immunofluorescence of Mena F/F (C) or α5 F/F (D) cells after infection with GFP or GFP-Cre adenovirus. Panels on the right show enlarged views of the boxed regions. Error bars indicate mean ± SEM. Bar, 10 µm.

    Techniques Used: Expressing, Western Blot, Isolation, Mouse Assay, Infection, Real-time Polymerase Chain Reaction, Immunofluorescence

    13) Product Images from "Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway †"

    Article Title: Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway †

    Journal: Biochemistry

    doi: 10.1021/bi900621w

    The WD domain of Vps15 is sufficient but not necessary to bind Atg14 Detergent-solubilized extracts (Total) from cells expressing the indicated Flag fusion proteins and Atg14 fused to a triple Myc epitope were incubated with Flag resin, eluted with 3X Flag peptide (Flag IP), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.
    Figure Legend Snippet: The WD domain of Vps15 is sufficient but not necessary to bind Atg14 Detergent-solubilized extracts (Total) from cells expressing the indicated Flag fusion proteins and Atg14 fused to a triple Myc epitope were incubated with Flag resin, eluted with 3X Flag peptide (Flag IP), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.

    Techniques Used: Expressing, Incubation, SDS Page, Immunoprecipitation

    The WD domain of Vps15 is sufficient but not necessary to bind Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Flag fusion proteins and Gpa1 fused to Myc were incubated with Flag resin, eluted with 3X Flag peptide (Flag IP), resolved by 7.5% or 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). C-terminally tagged forms of (a) and (b) did not express. IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.
    Figure Legend Snippet: The WD domain of Vps15 is sufficient but not necessary to bind Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Flag fusion proteins and Gpa1 fused to Myc were incubated with Flag resin, eluted with 3X Flag peptide (Flag IP), resolved by 7.5% or 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). C-terminally tagged forms of (a) and (b) did not express. IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.

    Techniques Used: Expressing, Incubation, SDS Page, Immunoprecipitation

    Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1 (A) Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Myc, GST, Ste4 and G6PDH (Load control). (B) Detergent-solubilized extracts from cells expressing the indicated Myc fusion proteins and Gpa1 fused to GST were lysed in the presence of either GDP or GDP-AlF 4 - , incubated with glutathione-Sepharose resin, eluted with glutathione, and analyzed by immunoblotting with antibodies against Myc, GST, Ste 4, and G6PDH. PD, pulldown. GST, glutathione S-transferase. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.
    Figure Legend Snippet: Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1 (A) Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Myc, GST, Ste4 and G6PDH (Load control). (B) Detergent-solubilized extracts from cells expressing the indicated Myc fusion proteins and Gpa1 fused to GST were lysed in the presence of either GDP or GDP-AlF 4 - , incubated with glutathione-Sepharose resin, eluted with glutathione, and analyzed by immunoblotting with antibodies against Myc, GST, Ste 4, and G6PDH. PD, pulldown. GST, glutathione S-transferase. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.

    Techniques Used: Expressing, Incubation, SDS Page

    Arg-1261 is necessary for the WD domain of Vps15 to bind efficiently to Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD, pulldown), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GST, Myc and G6PDH (Load control). PD, pulldown. GST, glutathione S-transferase. WD, WD domain. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.
    Figure Legend Snippet: Arg-1261 is necessary for the WD domain of Vps15 to bind efficiently to Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD, pulldown), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GST, Myc and G6PDH (Load control). PD, pulldown. GST, glutathione S-transferase. WD, WD domain. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.

    Techniques Used: Expressing, Incubation, SDS Page

    Atg14 is not necessary to mediate the interaction of Vps15 and Gpa1 Detergent-solubilized extracts from wildtype (WT) and atg14 Δ mutant cells expressing the indicated Flag fusion proteins and Gpa1 fused to a Myc epitope were incubated with Flag resin, eluted with 3X Flag peptide, resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.
    Figure Legend Snippet: Atg14 is not necessary to mediate the interaction of Vps15 and Gpa1 Detergent-solubilized extracts from wildtype (WT) and atg14 Δ mutant cells expressing the indicated Flag fusion proteins and Gpa1 fused to a Myc epitope were incubated with Flag resin, eluted with 3X Flag peptide, resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.

    Techniques Used: Mutagenesis, Expressing, Incubation, SDS Page, Immunoprecipitation

    The conserved Arg-1261 is not necessary for G protein signaling at the endosome (A) Wildtype (WT), vps15 Δ, and VPS15 R1261A (Vps15 RA ) strains were treated with 3μM α-mating factor (MF) for 30 minutes and analyzed by immunoblotting using antibodies against p44/p42 and G6PDH (Load control). (B) The same samples as in panel (A) analyzed using antibodies against carboxypeptidase Y (CPY) and G6PDH. (C) The same strains were transformed with a plasmid containing a pheromone-inducible FUS1-lacZ reporter; transcriptional activation was measured by monitoring β-galactosidase activity in response to pheromone. (D) The same strains were plated and treated with 45 micrograms of α factor to induce cell division arrest. α-MF, alpha mating factor. RA, Arg-1261 to Ala.
    Figure Legend Snippet: The conserved Arg-1261 is not necessary for G protein signaling at the endosome (A) Wildtype (WT), vps15 Δ, and VPS15 R1261A (Vps15 RA ) strains were treated with 3μM α-mating factor (MF) for 30 minutes and analyzed by immunoblotting using antibodies against p44/p42 and G6PDH (Load control). (B) The same samples as in panel (A) analyzed using antibodies against carboxypeptidase Y (CPY) and G6PDH. (C) The same strains were transformed with a plasmid containing a pheromone-inducible FUS1-lacZ reporter; transcriptional activation was measured by monitoring β-galactosidase activity in response to pheromone. (D) The same strains were plated and treated with 45 micrograms of α factor to induce cell division arrest. α-MF, alpha mating factor. RA, Arg-1261 to Ala.

    Techniques Used: Transformation Assay, Plasmid Preparation, Activation Assay, Activity Assay

    14) Product Images from "A Highly Conserved Leucine in Mammarenavirus Matrix Z Protein Is Required for Z Interaction with the Virus L Polymerase and Z Stability in Cells Harboring an Active Viral Ribonucleoprotein"

    Article Title: A Highly Conserved Leucine in Mammarenavirus Matrix Z Protein Is Required for Z Interaction with the Virus L Polymerase and Z Stability in Cells Harboring an Active Viral Ribonucleoprotein

    Journal: Journal of Virology

    doi: 10.1128/JVI.02256-17

    Subcellular distribution of the mutant Z proteins in the presence or absence of an active vRNP. 293T cells seeded on coverslips in a 24-well plate (1 × 10 5 cells/well) and cultured overnight were transfected with 25 ng of plasmid expressing WT or the indicated mutant Z proteins together with (B) or without (A) plasmids required for intracellular reconstruction of an active vRNP (vRNP) (pT7-MG/CAT, pC-T7pol, pC-L, and pC-NP). Forty-eight hours later, transfected cells were fixed and Strep-tagged Z proteins and HA-tagged NP expression detected by IF using a mouse monoclonal antibody to Strep and a rabbit polyclonal antibody to HA, respectively. Nuclei were detected by DAPI staining. Stained cells were analyzed with a confocal microscope.
    Figure Legend Snippet: Subcellular distribution of the mutant Z proteins in the presence or absence of an active vRNP. 293T cells seeded on coverslips in a 24-well plate (1 × 10 5 cells/well) and cultured overnight were transfected with 25 ng of plasmid expressing WT or the indicated mutant Z proteins together with (B) or without (A) plasmids required for intracellular reconstruction of an active vRNP (vRNP) (pT7-MG/CAT, pC-T7pol, pC-L, and pC-NP). Forty-eight hours later, transfected cells were fixed and Strep-tagged Z proteins and HA-tagged NP expression detected by IF using a mouse monoclonal antibody to Strep and a rabbit polyclonal antibody to HA, respectively. Nuclei were detected by DAPI staining. Stained cells were analyzed with a confocal microscope.

    Techniques Used: Mutagenesis, Cell Culture, Transfection, Plasmid Preparation, Expressing, Staining, Microscopy

    15) Product Images from "Targeted Deletion of Kcne2 Impairs HCN Channel Function in Mouse Thalamocortical Circuits"

    Article Title: Targeted Deletion of Kcne2 Impairs HCN Channel Function in Mouse Thalamocortical Circuits

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042756

    Kcne2 deletion down-regulates HCN1 and HCN2 protein expression in the brain. A , Exemplar chemiluminescence signals from western blots of whole brain lysates from Kcne2 +/+ and Kcne2 −/− mice, normalized to total protein concentration and probed with antibodies raised against HCN1, HCN2, HCN4 or GAPDH, as indicated. B , Mean chemiluminescence intensities for bands corresponding to known molecular weights for HCN1, HCN2, HCN4 and GAPDH from blots as in panel A , n = 3–4 mice per genotype. The cumulative GAPDH data on the left were obtained concomitantly with the HCN1 and HCN2 samples while the comparable GAPDH data on the right were obtained concomitantly with the HCN4 samples. *Significant difference between genotypes at 95% confidence interval. Error bars indicate SEM.
    Figure Legend Snippet: Kcne2 deletion down-regulates HCN1 and HCN2 protein expression in the brain. A , Exemplar chemiluminescence signals from western blots of whole brain lysates from Kcne2 +/+ and Kcne2 −/− mice, normalized to total protein concentration and probed with antibodies raised against HCN1, HCN2, HCN4 or GAPDH, as indicated. B , Mean chemiluminescence intensities for bands corresponding to known molecular weights for HCN1, HCN2, HCN4 and GAPDH from blots as in panel A , n = 3–4 mice per genotype. The cumulative GAPDH data on the left were obtained concomitantly with the HCN1 and HCN2 samples while the comparable GAPDH data on the right were obtained concomitantly with the HCN4 samples. *Significant difference between genotypes at 95% confidence interval. Error bars indicate SEM.

    Techniques Used: Expressing, Western Blot, Mouse Assay, Protein Concentration

    16) Product Images from "NMD3 Encodes an Essential Cytoplasmic Protein Required for Stable 60S Ribosomal Subunits in Saccharomyces cerevisiae"

    Article Title: NMD3 Encodes an Essential Cytoplasmic Protein Required for Stable 60S Ribosomal Subunits in Saccharomyces cerevisiae

    Journal: Molecular and Cellular Biology

    doi:

    Cell fractionation. Cells of strain CH1305 and containing a c-Myc-tagged NMD3 on the centromeric plasmid pAJ153 were fractionated into nuclear and cytoplasmic fractions. Protein from 10 μl each of total cell lysate (T), nuclear fraction (N), and cytoplasmic fraction (C) were separated by SDS-PAGE on 8% polyacrylamide gels. The separated proteins were transferred to nitrocellulose membrane, and Western blotting was performed with anti-c-Myc monoclonal antibody and anti-Topo II and G6PDH polyclonal antibodies.
    Figure Legend Snippet: Cell fractionation. Cells of strain CH1305 and containing a c-Myc-tagged NMD3 on the centromeric plasmid pAJ153 were fractionated into nuclear and cytoplasmic fractions. Protein from 10 μl each of total cell lysate (T), nuclear fraction (N), and cytoplasmic fraction (C) were separated by SDS-PAGE on 8% polyacrylamide gels. The separated proteins were transferred to nitrocellulose membrane, and Western blotting was performed with anti-c-Myc monoclonal antibody and anti-Topo II and G6PDH polyclonal antibodies.

    Techniques Used: Cell Fractionation, Plasmid Preparation, SDS Page, Western Blot

    17) Product Images from "Retinol dehydrogenase 13 protects the mouse retina from acute light damage"

    Article Title: Retinol dehydrogenase 13 protects the mouse retina from acute light damage

    Journal: Molecular Vision

    doi:

    Full-field electroretinogram responses and apoptosis detection in Rdh13 +/+ and Rdh13 −/− mice. A : The scotopic electroretinogram responses of Rdh13 +/+ and Rdh13 −/− mice at 10 months of age were recorded. B : The amplitudes of a- and b-waves for either genotype was plotted as the mean±SD (n=5, each group), *: p > 0.05. C : The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining showed that there was no obvious apoptosis in both Rdh13 +/+ and Rdh13 −/− mice at 10 months of age. D : Apoptosis genes expression in Rdh13 +/+ , Rdh13 +/− , and Rdh13 −/− mice was detected by western blots. There was no increased expression level of apoptosis genes in all genotypes, which was in accordance with the result of TUNEL.TNF-α, tumor necrosis factor alpha; Fas, TNF receptor superfamily member 6; Bax, B-cell lymphoma 2-associated X protein; P65, nuclear factor-kappa B P65; RPE, retinal pigment epithelia; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
    Figure Legend Snippet: Full-field electroretinogram responses and apoptosis detection in Rdh13 +/+ and Rdh13 −/− mice. A : The scotopic electroretinogram responses of Rdh13 +/+ and Rdh13 −/− mice at 10 months of age were recorded. B : The amplitudes of a- and b-waves for either genotype was plotted as the mean±SD (n=5, each group), *: p > 0.05. C : The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining showed that there was no obvious apoptosis in both Rdh13 +/+ and Rdh13 −/− mice at 10 months of age. D : Apoptosis genes expression in Rdh13 +/+ , Rdh13 +/− , and Rdh13 −/− mice was detected by western blots. There was no increased expression level of apoptosis genes in all genotypes, which was in accordance with the result of TUNEL.TNF-α, tumor necrosis factor alpha; Fas, TNF receptor superfamily member 6; Bax, B-cell lymphoma 2-associated X protein; P65, nuclear factor-kappa B P65; RPE, retinal pigment epithelia; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Techniques Used: Mouse Assay, TUNEL Assay, Staining, Expressing, Western Blot

    18) Product Images from "DYSLIPIDEMIA IS A MAJOR FACTOR IN STEM CELL DAMAGE INDUCED BY UNCONTROLLED LONG-TERM TYPE 2 DIABETES AND OBESITY IN THE RAT, AS SUGGESTED BY THE EFFECTS ON STEM CELL CULTURE"

    Article Title: DYSLIPIDEMIA IS A MAJOR FACTOR IN STEM CELL DAMAGE INDUCED BY UNCONTROLLED LONG-TERM TYPE 2 DIABETES AND OBESITY IN THE RAT, AS SUGGESTED BY THE EFFECTS ON STEM CELL CULTURE

    Journal: The journal of sexual medicine

    doi: 10.1016/j.jsxm.2018.09.019

    Incubation of ED-MDSC with increasing aged OZ serum caused a concentration dependent expression of myostatin protein, a pro-lipofibrotic and muscle mass inhibitor, but the incubation with increasing glucose exerted an opposite effect.
    Figure Legend Snippet: Incubation of ED-MDSC with increasing aged OZ serum caused a concentration dependent expression of myostatin protein, a pro-lipofibrotic and muscle mass inhibitor, but the incubation with increasing glucose exerted an opposite effect.

    Techniques Used: Incubation, Concentration Assay, Expressing

    19) Product Images from "Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis"

    Article Title: Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2010.20

    ( a and b ) Loss of Bax protects both CTLMC and MLMC from cytokine deprivation-induced apoptosis more potently than loss of Bak. Mast cells from wt, bax −/− , bak −/− and bax −/− bak −/− mice were deprived on cytokines for 86 or 40 h, respectively. Cell viability was analyzed by PI staining and FACS analysis. Data are presented as mean (±S.E.M.) of three independent experiments. ( c and d ) Survival of CTLMC and MLMC from wt, bim −/− puma −/− and vav-bcl-2 transgenic mice in absence of cytokines was also analyzed by PI staining and FACS analysis. Data are presented as mean (±S.D.) of at least three independent experiments. ( e ) A schematic picture illustrating the model of indirect activation of apoptosis involving the three groups of Bcl-2 family proteins controlling cell fate: pro-apoptotic BH3-only proteins (Bim and Puma), anti-apoptotic Bcl-2-like proteins (Bcl-2) and effector proteins (Bax/Bak)
    Figure Legend Snippet: ( a and b ) Loss of Bax protects both CTLMC and MLMC from cytokine deprivation-induced apoptosis more potently than loss of Bak. Mast cells from wt, bax −/− , bak −/− and bax −/− bak −/− mice were deprived on cytokines for 86 or 40 h, respectively. Cell viability was analyzed by PI staining and FACS analysis. Data are presented as mean (±S.E.M.) of three independent experiments. ( c and d ) Survival of CTLMC and MLMC from wt, bim −/− puma −/− and vav-bcl-2 transgenic mice in absence of cytokines was also analyzed by PI staining and FACS analysis. Data are presented as mean (±S.D.) of at least three independent experiments. ( e ) A schematic picture illustrating the model of indirect activation of apoptosis involving the three groups of Bcl-2 family proteins controlling cell fate: pro-apoptotic BH3-only proteins (Bim and Puma), anti-apoptotic Bcl-2-like proteins (Bcl-2) and effector proteins (Bax/Bak)

    Techniques Used: Mouse Assay, Staining, FACS, Transgenic Assay, Activation Assay

    ( a ) Expression of the receptors Kit and Fc ɛ RI on the cell surface of wt and bax −/− bak −/− CTLMC and MLMC as examined by flow cytometry. The colors black and gray represents wt and bax −/− bak −/− mast cells, respectively. A dashed line represents the staining with an isotype-matched control Ab, whereas the filled line represents staining with Abs for either Kit or Fc ɛ RI. β -hexosaminidase release upon Fc ɛ RI cross-linking of wt and bax −/− bak −/− (dko) CTLMC and MLMC, respectively ( b ). One representative of two independent experiments is presented
    Figure Legend Snippet: ( a ) Expression of the receptors Kit and Fc ɛ RI on the cell surface of wt and bax −/− bak −/− CTLMC and MLMC as examined by flow cytometry. The colors black and gray represents wt and bax −/− bak −/− mast cells, respectively. A dashed line represents the staining with an isotype-matched control Ab, whereas the filled line represents staining with Abs for either Kit or Fc ɛ RI. β -hexosaminidase release upon Fc ɛ RI cross-linking of wt and bax −/− bak −/− (dko) CTLMC and MLMC, respectively ( b ). One representative of two independent experiments is presented

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining

    ( a ) Expression of mRNA for Bcl-2 members was analyzed using RPA. Extracted mRNA, 2 μ g per sample, was analyzed using a mAPO-2 multi-probe template according to the RiboQuant System protocol. Presented is one representative of two independent experiments. ( b ) The expression levels of pro-survival bcl-2 family members were quantified in relation to control l32 and gapdh using a phosphor-imager device. Data is presented as fold difference compared with wild type of two different sets of RPA. ( c ) Both Bax and Bak proteins are expressed in wt CTLMC and control cells, mouse embryonic fibroblasts (MEF). Mast cells were either resting or activated by IgE receptor cross-linking for 5 h. One representative of three independent experiments is presented
    Figure Legend Snippet: ( a ) Expression of mRNA for Bcl-2 members was analyzed using RPA. Extracted mRNA, 2 μ g per sample, was analyzed using a mAPO-2 multi-probe template according to the RiboQuant System protocol. Presented is one representative of two independent experiments. ( b ) The expression levels of pro-survival bcl-2 family members were quantified in relation to control l32 and gapdh using a phosphor-imager device. Data is presented as fold difference compared with wild type of two different sets of RPA. ( c ) Both Bax and Bak proteins are expressed in wt CTLMC and control cells, mouse embryonic fibroblasts (MEF). Mast cells were either resting or activated by IgE receptor cross-linking for 5 h. One representative of three independent experiments is presented

    Techniques Used: Expressing, Recombinase Polymerase Amplification

    20) Product Images from "Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway"

    Article Title: Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway

    Journal: Blood

    doi: 10.1182/blood-2009-06-223719

    Inhibition of PDGF or LGL patient sera stimulation of NK leukemia cell line growth by blockade of PI3K/SFK or neutralizing antibody to PDGF-BB, respectively . (A-B) NKL cells were treated with rhPDGF-BB in presence of AG1296 at different concentrations
    Figure Legend Snippet: Inhibition of PDGF or LGL patient sera stimulation of NK leukemia cell line growth by blockade of PI3K/SFK or neutralizing antibody to PDGF-BB, respectively . (A-B) NKL cells were treated with rhPDGF-BB in presence of AG1296 at different concentrations

    Techniques Used: Inhibition

    21) Product Images from "Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina"

    Article Title: Peripheral Myelin Protein 22 Is in Complex with α6β4 Integrin, and Its Absence Alters the Schwann Cell Basal Lamina

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2618-05.2006

    Decreased levels of β4 integrin in nerves of PMP22-deficient mice. A–F , Cryosections of sciatic nerves from +/+ ( A , C , E ) and PMP22−/− ( B , D , F ) mice were immunostained with monoclonal rat (rt) anti-β4 integrin ( A , B ), anti-β1 integrin ( C , D ), or polyclonal rabbit (rb) anti-laminin (Lam) ( E , F ) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface ( A , C , arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the −/− samples ( B , D , arrows). In addition, when the images were collected at the same exposure times, the level of β4-like immunoreactivity was reduced (compare A , B ). Laminin was detected at the SC basal lamina in +/+ ( E , arrow) and −/− ( F ) nerves, with thickened basal lamina (arrowheads) and a tomaculum (open arrowhead) marked in the affected sample ( F ). Nonspecific rat ( A , B , inset) and rabbit ( E , F , inset) sera serve as controls for staining specificity. Scale bar, 5 μm. G , Sciatic nerve lysates (20 μg/lane) from P10 +/+, +/−, and −/− mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin and monoclonal rat anti-β1 integrin antibodies. The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kilodaltons.
    Figure Legend Snippet: Decreased levels of β4 integrin in nerves of PMP22-deficient mice. A–F , Cryosections of sciatic nerves from +/+ ( A , C , E ) and PMP22−/− ( B , D , F ) mice were immunostained with monoclonal rat (rt) anti-β4 integrin ( A , B ), anti-β1 integrin ( C , D ), or polyclonal rabbit (rb) anti-laminin (Lam) ( E , F ) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface ( A , C , arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the −/− samples ( B , D , arrows). In addition, when the images were collected at the same exposure times, the level of β4-like immunoreactivity was reduced (compare A , B ). Laminin was detected at the SC basal lamina in +/+ ( E , arrow) and −/− ( F ) nerves, with thickened basal lamina (arrowheads) and a tomaculum (open arrowhead) marked in the affected sample ( F ). Nonspecific rat ( A , B , inset) and rabbit ( E , F , inset) sera serve as controls for staining specificity. Scale bar, 5 μm. G , Sciatic nerve lysates (20 μg/lane) from P10 +/+, +/−, and −/− mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin and monoclonal rat anti-β1 integrin antibodies. The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kilodaltons.

    Techniques Used: Mouse Assay, Laser Capture Microdissection, Staining

    Coexpression of PMP22 and integrins during myelination. A , Sciatic nerve cryosections from 3-month-old +/+ mice were labeled with monoclonal rat anti-β4 and polyclonal rabbit anti-PMP22 antibodies and examined by confocal microscopy. The merged single plane image reveals the partial colocalization (merge, yellow) of β4 integrin (green) and PMP22 (red). Scale bar, 5 μm. Nonspecific rat (rt) and rabbit (rb) sera (bottom insets) serve as controls for staining specificity. B , Entire protein lysates of P1, P3, P8, and P21 sciatic nerves (10 μg/lane) from +/+ mice were analyzed with anti-PMP22 and anti-β4 integrin antibodies. The arrows indicate the migration of β4 integrin at ∼200 kDa (top) and of PMP22 at ∼22 kDa (bottom), whereas the arrowhead marks a nonspecific immunoreactive band. C , Cell-surface biotinylation of myelinating DRG–SC cocultures identifies β4 integrin and PMP22 in the avidin pull down (AP), from which actin is excluded. Total lysate (T) and agarose bead preclear (PC) fractions are also shown. Molecular mass is in kilodaltons. PMP, PMP22.
    Figure Legend Snippet: Coexpression of PMP22 and integrins during myelination. A , Sciatic nerve cryosections from 3-month-old +/+ mice were labeled with monoclonal rat anti-β4 and polyclonal rabbit anti-PMP22 antibodies and examined by confocal microscopy. The merged single plane image reveals the partial colocalization (merge, yellow) of β4 integrin (green) and PMP22 (red). Scale bar, 5 μm. Nonspecific rat (rt) and rabbit (rb) sera (bottom insets) serve as controls for staining specificity. B , Entire protein lysates of P1, P3, P8, and P21 sciatic nerves (10 μg/lane) from +/+ mice were analyzed with anti-PMP22 and anti-β4 integrin antibodies. The arrows indicate the migration of β4 integrin at ∼200 kDa (top) and of PMP22 at ∼22 kDa (bottom), whereas the arrowhead marks a nonspecific immunoreactive band. C , Cell-surface biotinylation of myelinating DRG–SC cocultures identifies β4 integrin and PMP22 in the avidin pull down (AP), from which actin is excluded. Total lysate (T) and agarose bead preclear (PC) fractions are also shown. Molecular mass is in kilodaltons. PMP, PMP22.

    Techniques Used: Mouse Assay, Labeling, Confocal Microscopy, Staining, Migration, Avidin-Biotin Assay

    PMP22 and β4 integrin are coimmunoprecipitated from clone A cells. A , B , Epitope (myc)-tagged hPMP22 was expressed in clone A cells, and samples were double immunolabeled with anti-β4 ( A ) and anti-myc ( B ) antibodies. C , As the merged image reveals, hPMP22 is targeted to the β4 integrin-positive plasma membrane of the cells ( A–C , arrows). Scale bar, 10 μm. D , Vector control and hPMP-myc-expressing clone A cells were lysed and processed for IP with the indicated antibodies. The precipitates were subsequently probed for the marked proteins by Western blot. The arrows on the right indicate the overexpressed glycosylated PMP22 (∼22 kDa) and the endogenous β4 integrin (∼200 kDa). The arrowheads point at the position of the deglycosylated ∼18 and ∼190 kDa forms. Molecular mass is in kilodaltons. T, Total lysate; PC, preclear.
    Figure Legend Snippet: PMP22 and β4 integrin are coimmunoprecipitated from clone A cells. A , B , Epitope (myc)-tagged hPMP22 was expressed in clone A cells, and samples were double immunolabeled with anti-β4 ( A ) and anti-myc ( B ) antibodies. C , As the merged image reveals, hPMP22 is targeted to the β4 integrin-positive plasma membrane of the cells ( A–C , arrows). Scale bar, 10 μm. D , Vector control and hPMP-myc-expressing clone A cells were lysed and processed for IP with the indicated antibodies. The precipitates were subsequently probed for the marked proteins by Western blot. The arrows on the right indicate the overexpressed glycosylated PMP22 (∼22 kDa) and the endogenous β4 integrin (∼200 kDa). The arrowheads point at the position of the deglycosylated ∼18 and ∼190 kDa forms. Molecular mass is in kilodaltons. T, Total lysate; PC, preclear.

    Techniques Used: Immunolabeling, Plasmid Preparation, Expressing, Western Blot

    PMP22 is in a complex with α6β4 integrin and laminin. Sciatic nerve lysates (T lanes) from P21 +/+ mice were processed for IP, after preclearing (PC lanes) with nonspecific Igs of the appropriate isotype. Lysates were incubated with polyclonal rabbit anti-β4 integrin ( A , left, C ), anti-PMP22 ( A , right), monoclonal rat anti-α6 integrin ( B , left), or polyclonal rabbit anti-laminin ( B , right) antibodies, and captured immunoprecipitates were probed for the indicated proteins ( A–C ) as designated at the right of each blot. On reprobes (marked with asterisks), after stripping the membranes, the anti-β4 integrin and anti-laminin antibodies did not work efficiently when 1 μg/lane total (T) nerve protein was analyzed. IP with anti-β4 integrin on nerve lysates of homozygous PMP22-deficient mice is shown as a negative control ( C ). Molecular mass is in kilodaltons. PMP, PMP22; Lam, laminin; βDys, β-dystroglycan.
    Figure Legend Snippet: PMP22 is in a complex with α6β4 integrin and laminin. Sciatic nerve lysates (T lanes) from P21 +/+ mice were processed for IP, after preclearing (PC lanes) with nonspecific Igs of the appropriate isotype. Lysates were incubated with polyclonal rabbit anti-β4 integrin ( A , left, C ), anti-PMP22 ( A , right), monoclonal rat anti-α6 integrin ( B , left), or polyclonal rabbit anti-laminin ( B , right) antibodies, and captured immunoprecipitates were probed for the indicated proteins ( A–C ) as designated at the right of each blot. On reprobes (marked with asterisks), after stripping the membranes, the anti-β4 integrin and anti-laminin antibodies did not work efficiently when 1 μg/lane total (T) nerve protein was analyzed. IP with anti-β4 integrin on nerve lysates of homozygous PMP22-deficient mice is shown as a negative control ( C ). Molecular mass is in kilodaltons. PMP, PMP22; Lam, laminin; βDys, β-dystroglycan.

    Techniques Used: Mouse Assay, Incubation, Stripping Membranes, Negative Control, Laser Capture Microdissection

    22) Product Images from "Deubiquitinase inhibitor PR-619 reduces Smad4 expression and suppresses renal fibrosis in mice with unilateral ureteral obstruction"

    Article Title: Deubiquitinase inhibitor PR-619 reduces Smad4 expression and suppresses renal fibrosis in mice with unilateral ureteral obstruction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202409

    PR-619 suppresses UUO-induced ECM deposition. The same samples in Fig 2 were used to examine collagen 1, collagen 3, and fibronectin as ECM proteins. (A) Collagen 1, collagen 3, and fibronectin mRNA levels were determined by qRT-PCR in mice with UUO with or without PR-619 administration. GAPDH was used as an internal control. (B) Typical western blot analysis demonstrating the level of fibronectin protein expression. The graph shows the expression level quantified by densitometry and normalized to GAPDH. (C) Representative images showing immunostaining for collagen 1, collagen 3, and fibronectin. Quantification is shown in the right panel. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. * P
    Figure Legend Snippet: PR-619 suppresses UUO-induced ECM deposition. The same samples in Fig 2 were used to examine collagen 1, collagen 3, and fibronectin as ECM proteins. (A) Collagen 1, collagen 3, and fibronectin mRNA levels were determined by qRT-PCR in mice with UUO with or without PR-619 administration. GAPDH was used as an internal control. (B) Typical western blot analysis demonstrating the level of fibronectin protein expression. The graph shows the expression level quantified by densitometry and normalized to GAPDH. (C) Representative images showing immunostaining for collagen 1, collagen 3, and fibronectin. Quantification is shown in the right panel. Values are expressed as the mean ± SD. Statistical analysis was performed using ANOVA followed by Tukey’s post hoc test. * P

    Techniques Used: Quantitative RT-PCR, Mouse Assay, Western Blot, Expressing, Immunostaining

    23) Product Images from "Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation"

    Article Title: Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation

    Journal: Journal of Virology

    doi: 10.1128/JVI.01690-17

    Inhibition of HA expression by endogenous EDEM1, EDEM2, and ERManI. (A) WT 293T cells were transfected with EDEM1, EDEM2, and ERManI expression vectors plus a lentiviral vector expressing the specific shRNAs or a scrambled shRNA as a control (ctrl). Protein expression was analyzed by Western blotting. (B) ΔΔ 293T cells were transfected with an H5 or HIV-1 proviral vector plus a lentiviral vector expressing the indicated shRNAs. Protein expression was analyzed by Western blotting. (C) WT A549 and 293T cells were stably transduced with a lentiviral vector expressing the indicated shRNAs, and HA expression in these cells was analyzed by Western blotting.
    Figure Legend Snippet: Inhibition of HA expression by endogenous EDEM1, EDEM2, and ERManI. (A) WT 293T cells were transfected with EDEM1, EDEM2, and ERManI expression vectors plus a lentiviral vector expressing the specific shRNAs or a scrambled shRNA as a control (ctrl). Protein expression was analyzed by Western blotting. (B) ΔΔ 293T cells were transfected with an H5 or HIV-1 proviral vector plus a lentiviral vector expressing the indicated shRNAs. Protein expression was analyzed by Western blotting. (C) WT A549 and 293T cells were stably transduced with a lentiviral vector expressing the indicated shRNAs, and HA expression in these cells was analyzed by Western blotting.

    Techniques Used: Inhibition, Expressing, Transfection, Plasmid Preparation, shRNA, Western Blot, Stable Transfection, Transduction

    Inhibition of HA expression by EDEM1, EDEM2, and ERManI. (A) 293T cells were transfected with an H5 expression vector and a vector expressing EDEM1, EDEM2, or ERManI with a C-terminal FLAG tag. After 48 h, cells were lysed and analyzed via Western blotting. APOBEC3A (A3A) was used as a control. (B) HA expression on Western blots shown in panel A was quantified with ImageJ and is presented as relative values. Results are displayed as the means ± SD ( n = 3). *, P
    Figure Legend Snippet: Inhibition of HA expression by EDEM1, EDEM2, and ERManI. (A) 293T cells were transfected with an H5 expression vector and a vector expressing EDEM1, EDEM2, or ERManI with a C-terminal FLAG tag. After 48 h, cells were lysed and analyzed via Western blotting. APOBEC3A (A3A) was used as a control. (B) HA expression on Western blots shown in panel A was quantified with ImageJ and is presented as relative values. Results are displayed as the means ± SD ( n = 3). *, P

    Techniques Used: Inhibition, Expressing, Transfection, Plasmid Preparation, FLAG-tag, Western Blot

    Inhibition of IAV replication by EDEM1, EDEM2, and ERManI. (A) Stable A549 cell lines expressing the indicated shRNAs were infected with H1N1 A/WSN/33 viruses at an MOI of 0.5. Viral supernatants were sampled at the specified time points. Viral titers were determined by a hemagglutination assay using turkey red blood cells and plaque-forming cell assay after infecting MDCK cells. Results are displayed as means ± SD ( n = 2). *, P
    Figure Legend Snippet: Inhibition of IAV replication by EDEM1, EDEM2, and ERManI. (A) Stable A549 cell lines expressing the indicated shRNAs were infected with H1N1 A/WSN/33 viruses at an MOI of 0.5. Viral supernatants were sampled at the specified time points. Viral titers were determined by a hemagglutination assay using turkey red blood cells and plaque-forming cell assay after infecting MDCK cells. Results are displayed as means ± SD ( n = 2). *, P

    Techniques Used: Inhibition, Expressing, Infection, Hemagglutination Assay

    Requirement of mannosidase activity for HA inhibition. (A) 293T cells were transfected with an H5 vector and the indicated EDEM and ERManI expression vectors. After 24 h, cells were treated with kifunensine (5 μM), lactacystin (25 μM), or MG132 (25 μM) for 6 h, and protein expression was analyzed by Western blotting. (B) 293T cells were transfected with an H5 vector and the indicated EDEM and ERManI WT or catalytic mutant expression vector, and protein expression was analyzed by Western blotting. (C) ERManI-KO 293T cells were transfected with an H5 vector and the indicated EDEM WT or mutant expression vector, and protein expression was analyzed by Western blotting. (D) The relative expression of ERManI, EDEM1, EDEM2, and EDEM3 in the human lung epithelial cell line A549 was determined by real-time qPCR. Results are displayed as means ± SEM ( n = 3). *, P
    Figure Legend Snippet: Requirement of mannosidase activity for HA inhibition. (A) 293T cells were transfected with an H5 vector and the indicated EDEM and ERManI expression vectors. After 24 h, cells were treated with kifunensine (5 μM), lactacystin (25 μM), or MG132 (25 μM) for 6 h, and protein expression was analyzed by Western blotting. (B) 293T cells were transfected with an H5 vector and the indicated EDEM and ERManI WT or catalytic mutant expression vector, and protein expression was analyzed by Western blotting. (C) ERManI-KO 293T cells were transfected with an H5 vector and the indicated EDEM WT or mutant expression vector, and protein expression was analyzed by Western blotting. (D) The relative expression of ERManI, EDEM1, EDEM2, and EDEM3 in the human lung epithelial cell line A549 was determined by real-time qPCR. Results are displayed as means ± SEM ( n = 3). *, P

    Techniques Used: Activity Assay, Inhibition, Transfection, Plasmid Preparation, Expressing, Western Blot, Mutagenesis, Real-time Polymerase Chain Reaction

    24) Product Images from "Novel role of the muskelin-RanBP9 complex as a nucleocytoplasmic mediator of cell morphology regulation"

    Article Title: Novel role of the muskelin-RanBP9 complex as a nucleocytoplasmic mediator of cell morphology regulation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200801133

    The LisH motif of muskelin has nuclear localization activity. (A) Sequence Logos for the consensus LisH motif of muskelin (LisH MK ), derived by alignment of LisH motifs from 12 muskelin sequences from vertebrates, insects, and fungi, and the general consensus for a LisH motif, derived by alignment of 120 LisH motifs from vertebrates, insects, and fungi in the SMART database. Amino acids are shown in single letter code. The total height at each position is a measure of the strength of conservation at that position. Letter heights display the relative conservation of individual amino acids at that position. (B) Vinculin reporter constructs prepared without or with C-terminal fusion of GFP or LisH MK were expressed in COS-7 cells. Immunoblots of 5 and 20 μg of each whole cell extract demonstrate equivalent expression of vinculin, vinculin-LisH MK , and vinculin-LisHAA and higher expression of vinculin-GFP. Arrows indicate the ectopically expressed vinculins and asterisks indicate the endogenous vinculin. (C) LisH MK mediates nuclear localization of vinculin. Confocal xy or xz images of anti-vinculin–stained COS-7 cells expressing the indicated constructs. Bars, 10 μm. (D) Quantification of subcellular localizations of vinculin reporter proteins. Each column represents the mean from three independent experiments. Error bars indicate SEM. At least 500 cells were scored for each construct. (E) Immunoblot demonstrates equivalent expression of GFP-MK and GFP-MKΔC35, without or with point mutation of LisH MK , in COS-7 cells. Molecular mass markers are shown in kilodaltons. (F) Merged confocal images of representative localizations of the indicated GFP-tagged proteins (green) and DAPI-stained nuclei (blue). Bar, 10 μm. (G) Quantification of subcellular localizations. Each column represents the mean from three to five independent experiments. Error bars indicate SEM. At least 500 cells were scored for each construct.
    Figure Legend Snippet: The LisH motif of muskelin has nuclear localization activity. (A) Sequence Logos for the consensus LisH motif of muskelin (LisH MK ), derived by alignment of LisH motifs from 12 muskelin sequences from vertebrates, insects, and fungi, and the general consensus for a LisH motif, derived by alignment of 120 LisH motifs from vertebrates, insects, and fungi in the SMART database. Amino acids are shown in single letter code. The total height at each position is a measure of the strength of conservation at that position. Letter heights display the relative conservation of individual amino acids at that position. (B) Vinculin reporter constructs prepared without or with C-terminal fusion of GFP or LisH MK were expressed in COS-7 cells. Immunoblots of 5 and 20 μg of each whole cell extract demonstrate equivalent expression of vinculin, vinculin-LisH MK , and vinculin-LisHAA and higher expression of vinculin-GFP. Arrows indicate the ectopically expressed vinculins and asterisks indicate the endogenous vinculin. (C) LisH MK mediates nuclear localization of vinculin. Confocal xy or xz images of anti-vinculin–stained COS-7 cells expressing the indicated constructs. Bars, 10 μm. (D) Quantification of subcellular localizations of vinculin reporter proteins. Each column represents the mean from three independent experiments. Error bars indicate SEM. At least 500 cells were scored for each construct. (E) Immunoblot demonstrates equivalent expression of GFP-MK and GFP-MKΔC35, without or with point mutation of LisH MK , in COS-7 cells. Molecular mass markers are shown in kilodaltons. (F) Merged confocal images of representative localizations of the indicated GFP-tagged proteins (green) and DAPI-stained nuclei (blue). Bar, 10 μm. (G) Quantification of subcellular localizations. Each column represents the mean from three to five independent experiments. Error bars indicate SEM. At least 500 cells were scored for each construct.

    Techniques Used: Activity Assay, Sequencing, Derivative Assay, Construct, Western Blot, Expressing, Staining, Mutagenesis

    25) Product Images from "Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway"

    Article Title: Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.4870

    JNK inhibitor, SP600125, reverses the viability and apoptosis of suspended HUVECs induced by DHA. (A) Representative immunoblots of p-JNK and JNK in suspended HUVECs treated with DHA and SP600125. (B) Percentage of viable cells from suspended HUVECs treated with DHA and SP600125; n=4. (C) Representative images of flow cytometry analyses of Annexin V/PI-staining in suspended HUVECs treated with DHA and SP600125. DHA, dihydroartemisinin; JNK, c-Jun N-terminal kinase; p-JNK, phosphorylated-JNK; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell; n.s., non-significant; PI, propidium iodide; FITC, fluorescein isothiocyanate.
    Figure Legend Snippet: JNK inhibitor, SP600125, reverses the viability and apoptosis of suspended HUVECs induced by DHA. (A) Representative immunoblots of p-JNK and JNK in suspended HUVECs treated with DHA and SP600125. (B) Percentage of viable cells from suspended HUVECs treated with DHA and SP600125; n=4. (C) Representative images of flow cytometry analyses of Annexin V/PI-staining in suspended HUVECs treated with DHA and SP600125. DHA, dihydroartemisinin; JNK, c-Jun N-terminal kinase; p-JNK, phosphorylated-JNK; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell; n.s., non-significant; PI, propidium iodide; FITC, fluorescein isothiocyanate.

    Techniques Used: Western Blot, Flow Cytometry, Cytometry, Staining

    26) Product Images from "Defective in Mitotic Arrest 1 (Dma1) Ubiquitin Ligase Controls G1 Cyclin Degradation *"

    Article Title: Defective in Mitotic Arrest 1 (Dma1) Ubiquitin Ligase Controls G1 Cyclin Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.426593

    Cln2 and Pcl1 disappear with different kinetics. Strain YSH98 (double-tagged: CLN2 -TAP and PCL1 -TAP) was synchronized at G 1 with α-factor and released in YPD at time 0. Samples were collected at the indicated times and then subjected to several analyses. A, Cln2 and Pcl1 protein levels were analyzed by immunoblotting using monoclonal antibodies against TAP-tag; Glu-6-PDH ( G6PDH ) detection was used as a loading control. B , quantification of panel A . Data ± S.D. from 3 independent experiments are shown. C, flow cytometry analysis of DNA content of samples from panel A. D , RNA levels of Cln2 and Pcl1 of samples from panel A . Average from two independent experiments is shown.
    Figure Legend Snippet: Cln2 and Pcl1 disappear with different kinetics. Strain YSH98 (double-tagged: CLN2 -TAP and PCL1 -TAP) was synchronized at G 1 with α-factor and released in YPD at time 0. Samples were collected at the indicated times and then subjected to several analyses. A, Cln2 and Pcl1 protein levels were analyzed by immunoblotting using monoclonal antibodies against TAP-tag; Glu-6-PDH ( G6PDH ) detection was used as a loading control. B , quantification of panel A . Data ± S.D. from 3 independent experiments are shown. C, flow cytometry analysis of DNA content of samples from panel A. D , RNA levels of Cln2 and Pcl1 of samples from panel A . Average from two independent experiments is shown.

    Techniques Used: Flow Cytometry, Cytometry

    Pcl1 has a putative docking site for Dma1. A , the integrity of the DDD region is essential to maintaining Pcl1 instability in vivo . Different alleles of Pcl1 were expressed in wild type cells from a centromeric plasmid under its own promoter: Pcl1 wild type, Pcl1 with a deletion in the DDD region ( DDD Δ), and Pcl1 with mutations V31N and P33N ( DDD *). All these strains were grown exponentially in rich medium and, at time 0, cycloheximide was added to the medium. Samples were taken at the indicated times and analyzed by immunoblotting using monoclonal antibodies to determine Pcl1-TAP levels. Glu-6-PDH ( G6PDH ) detection was used as a loading control. B, quantification of panel A . Data ± S.D. from 3 independent experiments are shown. **, p > 0.01 versus WT. C, pcl1 DDD Δ is less ubiquitinated in vitro than Pcl1. Ubiquitination assays were done by in vitro reconstitution of E1-Mms2-Ubc13-Dma1 complexes (see “Experimental Procedures”). The ubiquitination levels of Pcl1 or pcl1 DDD Δ are shown. The reaction was started at time 0 by addition of ATP and finished at 3 h. Samples were taken and analyzed for ubiquitination levels by immunoblotting using α-Ub antibody. D, ) of the DDD regions present in several Dma-interacting proteins. E, T-Coffee alignment between the N-terminal region of cyclins Pcl1 and Cln2. Cln2 presents a gap that exactly matches the proposed DDD region.
    Figure Legend Snippet: Pcl1 has a putative docking site for Dma1. A , the integrity of the DDD region is essential to maintaining Pcl1 instability in vivo . Different alleles of Pcl1 were expressed in wild type cells from a centromeric plasmid under its own promoter: Pcl1 wild type, Pcl1 with a deletion in the DDD region ( DDD Δ), and Pcl1 with mutations V31N and P33N ( DDD *). All these strains were grown exponentially in rich medium and, at time 0, cycloheximide was added to the medium. Samples were taken at the indicated times and analyzed by immunoblotting using monoclonal antibodies to determine Pcl1-TAP levels. Glu-6-PDH ( G6PDH ) detection was used as a loading control. B, quantification of panel A . Data ± S.D. from 3 independent experiments are shown. **, p > 0.01 versus WT. C, pcl1 DDD Δ is less ubiquitinated in vitro than Pcl1. Ubiquitination assays were done by in vitro reconstitution of E1-Mms2-Ubc13-Dma1 complexes (see “Experimental Procedures”). The ubiquitination levels of Pcl1 or pcl1 DDD Δ are shown. The reaction was started at time 0 by addition of ATP and finished at 3 h. Samples were taken and analyzed for ubiquitination levels by immunoblotting using α-Ub antibody. D, ) of the DDD regions present in several Dma-interacting proteins. E, T-Coffee alignment between the N-terminal region of cyclins Pcl1 and Cln2. Cln2 presents a gap that exactly matches the proposed DDD region.

    Techniques Used: In Vivo, Plasmid Preparation, In Vitro

    Pcl1 is more stable in the absence of Dma activity. A , Pcl1 levels increased in the presence of proteasomal inhibitors. Cells of strain YSH82 were incubated with either MG132 (a proteasome inhibitor) or a drug vehicle ( DMSO ). 60 min later, samples were taken and Pcl1-TAP protein levels were analyzed by immunoblotting using monoclonal antibodies against TAP-tag. Two different exposures of the immunoblot are shown. As in the rest of the panels, Glu-6-PDH ( G6PDH ) detection was used as a loading control. B , relative amounts of Pcl1 in wild type, grr1 Δ, and dma1 Δ dma2 Δ strains. Cells were grown exponentially in rich media, and Pcl1-TAP levels were detected by immunoblotting using monoclonal antibodies. C , Pcl1 stability measurements. The indicated strains were grown exponentially in rich media and cycloheximide was added to the medium at time 0. Samples were taken at the indicated times and analyzed for Pcl1-TAP levels by immunoblotting using monoclonal antibodies. D, quantification of panel C . Data ± S.D. from 3 independent experiments are shown. *, p > 0.05 versus WT. E, Pcl9 stability measurements were carried out as in panel C. F, quantification of panel E . Data ± S.D. from 3 independent experiments are shown.
    Figure Legend Snippet: Pcl1 is more stable in the absence of Dma activity. A , Pcl1 levels increased in the presence of proteasomal inhibitors. Cells of strain YSH82 were incubated with either MG132 (a proteasome inhibitor) or a drug vehicle ( DMSO ). 60 min later, samples were taken and Pcl1-TAP protein levels were analyzed by immunoblotting using monoclonal antibodies against TAP-tag. Two different exposures of the immunoblot are shown. As in the rest of the panels, Glu-6-PDH ( G6PDH ) detection was used as a loading control. B , relative amounts of Pcl1 in wild type, grr1 Δ, and dma1 Δ dma2 Δ strains. Cells were grown exponentially in rich media, and Pcl1-TAP levels were detected by immunoblotting using monoclonal antibodies. C , Pcl1 stability measurements. The indicated strains were grown exponentially in rich media and cycloheximide was added to the medium at time 0. Samples were taken at the indicated times and analyzed for Pcl1-TAP levels by immunoblotting using monoclonal antibodies. D, quantification of panel C . Data ± S.D. from 3 independent experiments are shown. *, p > 0.05 versus WT. E, Pcl9 stability measurements were carried out as in panel C. F, quantification of panel E . Data ± S.D. from 3 independent experiments are shown.

    Techniques Used: Activity Assay, Incubation

    Pcl1 is destabilized by Pho85 activity. A , Pcl1 protein levels are higher in the pho85 Δ strain. Wild type and pho85 Δ cells were grown exponentially in YPD, and then Pcl1-TAP levels were determined using immunoblotting with monoclonal antibodies. Glu-6-PDH ( G6PDH ) detection was used as a loading control in each panel. B , Pcl1 is stabilized in the pho85 Δ strain. Wild type and pho85 Δ cells were grown exponentially in rich medium, and cycloheximide was added to the medium at time 0. Samples were taken at the indicated times, and analyzed for Pcl1-TAP levels by immunoblotting using monoclonal antibodies. C, quantification of panel B . Data ± S.D. from 3 independent experiments are shown. **, p > 0.01 versus WT. D, Pcl9 stability is also controlled by Pho85. Analyses were carried out as in panel B .
    Figure Legend Snippet: Pcl1 is destabilized by Pho85 activity. A , Pcl1 protein levels are higher in the pho85 Δ strain. Wild type and pho85 Δ cells were grown exponentially in YPD, and then Pcl1-TAP levels were determined using immunoblotting with monoclonal antibodies. Glu-6-PDH ( G6PDH ) detection was used as a loading control in each panel. B , Pcl1 is stabilized in the pho85 Δ strain. Wild type and pho85 Δ cells were grown exponentially in rich medium, and cycloheximide was added to the medium at time 0. Samples were taken at the indicated times, and analyzed for Pcl1-TAP levels by immunoblotting using monoclonal antibodies. C, quantification of panel B . Data ± S.D. from 3 independent experiments are shown. **, p > 0.01 versus WT. D, Pcl9 stability is also controlled by Pho85. Analyses were carried out as in panel B .

    Techniques Used: Activity Assay

    Pho85 and Dma1 activities are essential for controlling Pcl1 levels in the cell cycle. A, proposed model of Pcl1 targeting. Pho85 phosphorylates Pcl1 allowing the recognition by Dma1 that polyubiquitinates and targets Pcl1 for destruction. B, the DNA content from cells collected in the experiment shown in C was analyzed by flow cytometry at the indicated time points. C, Pcl1 requires Dma1 activity to be destabilized under physiological conditions. Wild type cells carrying a Pcl1 or pcl1 2A version expressed from a centromeric plasmid and dma1 Δ dma2 Δ cells expressing Pcl1, were synchronized with α-factor, and released in rich medium. Samples were collected at the indicated times and then subjected to several analyses. Glu-6-PDH levels were used as a loading control.
    Figure Legend Snippet: Pho85 and Dma1 activities are essential for controlling Pcl1 levels in the cell cycle. A, proposed model of Pcl1 targeting. Pho85 phosphorylates Pcl1 allowing the recognition by Dma1 that polyubiquitinates and targets Pcl1 for destruction. B, the DNA content from cells collected in the experiment shown in C was analyzed by flow cytometry at the indicated time points. C, Pcl1 requires Dma1 activity to be destabilized under physiological conditions. Wild type cells carrying a Pcl1 or pcl1 2A version expressed from a centromeric plasmid and dma1 Δ dma2 Δ cells expressing Pcl1, were synchronized with α-factor, and released in rich medium. Samples were collected at the indicated times and then subjected to several analyses. Glu-6-PDH levels were used as a loading control.

    Techniques Used: Flow Cytometry, Cytometry, Activity Assay, Plasmid Preparation, Expressing

    G 1 cyclin levels are controlled in response to nutrients. A, Pcl1 levels are controlled in response to nutrients. The strain YSH98 (double tagged: CLN2 -TAP and PCL1 -TAP) was synchronized at G 1 with α-factor. At time 0 the cells were released in either YPD or SD media. Samples were collected at the indicated times, and Cln2 and Pcl1 levels were analyzed by immunoblotting using monoclonal antibodies against TAP-tag. Glu-6-PDH ( G6PDH ) levels were used as a loading control. B , Dma1 levels are controlled in response to nutrients. Strain YPC708 was synchronized, sampled, and analyzed as described in panel A .
    Figure Legend Snippet: G 1 cyclin levels are controlled in response to nutrients. A, Pcl1 levels are controlled in response to nutrients. The strain YSH98 (double tagged: CLN2 -TAP and PCL1 -TAP) was synchronized at G 1 with α-factor. At time 0 the cells were released in either YPD or SD media. Samples were collected at the indicated times, and Cln2 and Pcl1 levels were analyzed by immunoblotting using monoclonal antibodies against TAP-tag. Glu-6-PDH ( G6PDH ) levels were used as a loading control. B , Dma1 levels are controlled in response to nutrients. Strain YPC708 was synchronized, sampled, and analyzed as described in panel A .

    Techniques Used:

    27) Product Images from "Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway †"

    Article Title: Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway †

    Journal: Biochemistry

    doi: 10.1021/bi900621w

    The WD domain of Vps15 is sufficient but not necessary to bind Atg14 Detergent-solubilized extracts (Total) from cells expressing the indicated Flag fusion proteins and Atg14 fused to a triple Myc epitope were incubated with Flag resin, eluted with 3X Flag peptide (Flag IP), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.
    Figure Legend Snippet: The WD domain of Vps15 is sufficient but not necessary to bind Atg14 Detergent-solubilized extracts (Total) from cells expressing the indicated Flag fusion proteins and Atg14 fused to a triple Myc epitope were incubated with Flag resin, eluted with 3X Flag peptide (Flag IP), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.

    Techniques Used: Expressing, Incubation, SDS Page, Immunoprecipitation

    The WD domain of Vps15 is sufficient but not necessary to bind Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Flag fusion proteins and Gpa1 fused to Myc were incubated with Flag resin, eluted with 3X Flag peptide (Flag IP), resolved by 7.5% or 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). C-terminally tagged forms of (a) and (b) did not express. IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.
    Figure Legend Snippet: The WD domain of Vps15 is sufficient but not necessary to bind Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Flag fusion proteins and Gpa1 fused to Myc were incubated with Flag resin, eluted with 3X Flag peptide (Flag IP), resolved by 7.5% or 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). C-terminally tagged forms of (a) and (b) did not express. IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.

    Techniques Used: Expressing, Incubation, SDS Page, Immunoprecipitation

    Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1 (A) Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Myc, GST, Ste4 and G6PDH (Load control). (B) Detergent-solubilized extracts from cells expressing the indicated Myc fusion proteins and Gpa1 fused to GST were lysed in the presence of either GDP or GDP-AlF 4 - , incubated with glutathione-Sepharose resin, eluted with glutathione, and analyzed by immunoblotting with antibodies against Myc, GST, Ste 4, and G6PDH. PD, pulldown. GST, glutathione S-transferase. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.
    Figure Legend Snippet: Arg-1261 is not necessary for larger truncations of Vps15 to bind to Gpa1 (A) Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Myc, GST, Ste4 and G6PDH (Load control). (B) Detergent-solubilized extracts from cells expressing the indicated Myc fusion proteins and Gpa1 fused to GST were lysed in the presence of either GDP or GDP-AlF 4 - , incubated with glutathione-Sepharose resin, eluted with glutathione, and analyzed by immunoblotting with antibodies against Myc, GST, Ste 4, and G6PDH. PD, pulldown. GST, glutathione S-transferase. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.

    Techniques Used: Expressing, Incubation, SDS Page

    Arg-1261 is necessary for the WD domain of Vps15 to bind efficiently to Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD, pulldown), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GST, Myc and G6PDH (Load control). PD, pulldown. GST, glutathione S-transferase. WD, WD domain. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.
    Figure Legend Snippet: Arg-1261 is necessary for the WD domain of Vps15 to bind efficiently to Gpa1 Detergent-solubilized extracts (Total) from cells expressing the indicated Myc fusion proteins and Gpa1 or Gpa2 fused to GST were incubated with glutathione-Sepharose resin, eluted with glutathione (GST PD, pulldown), resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GST, Myc and G6PDH (Load control). PD, pulldown. GST, glutathione S-transferase. WD, WD domain. RA, Arg-1261 to Ala. RK, Arg-1261 to Lys.

    Techniques Used: Expressing, Incubation, SDS Page

    Atg14 is not necessary to mediate the interaction of Vps15 and Gpa1 Detergent-solubilized extracts from wildtype (WT) and atg14 Δ mutant cells expressing the indicated Flag fusion proteins and Gpa1 fused to a Myc epitope were incubated with Flag resin, eluted with 3X Flag peptide, resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.
    Figure Legend Snippet: Atg14 is not necessary to mediate the interaction of Vps15 and Gpa1 Detergent-solubilized extracts from wildtype (WT) and atg14 Δ mutant cells expressing the indicated Flag fusion proteins and Gpa1 fused to a Myc epitope were incubated with Flag resin, eluted with 3X Flag peptide, resolved by 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Flag, Myc, and G6PDH (Load control). IP, immunoprecipitation. WD, WD domain. KD, kinase domain. ID, intermediate domain. *, indicates protein of interest.

    Techniques Used: Mutagenesis, Expressing, Incubation, SDS Page, Immunoprecipitation

    The conserved Arg-1261 is not necessary for G protein signaling at the endosome (A) Wildtype (WT), vps15 Δ, and VPS15 R1261A (Vps15 RA ) strains were treated with 3μM α-mating factor (MF) for 30 minutes and analyzed by immunoblotting using antibodies against p44/p42 and G6PDH (Load control). (B) The same samples as in panel (A) analyzed using antibodies against carboxypeptidase Y (CPY) and G6PDH. (C) The same strains were transformed with a plasmid containing a pheromone-inducible FUS1-lacZ reporter; transcriptional activation was measured by monitoring β-galactosidase activity in response to pheromone. (D) The same strains were plated and treated with 45 micrograms of α factor to induce cell division arrest. α-MF, alpha mating factor. RA, Arg-1261 to Ala.
    Figure Legend Snippet: The conserved Arg-1261 is not necessary for G protein signaling at the endosome (A) Wildtype (WT), vps15 Δ, and VPS15 R1261A (Vps15 RA ) strains were treated with 3μM α-mating factor (MF) for 30 minutes and analyzed by immunoblotting using antibodies against p44/p42 and G6PDH (Load control). (B) The same samples as in panel (A) analyzed using antibodies against carboxypeptidase Y (CPY) and G6PDH. (C) The same strains were transformed with a plasmid containing a pheromone-inducible FUS1-lacZ reporter; transcriptional activation was measured by monitoring β-galactosidase activity in response to pheromone. (D) The same strains were plated and treated with 45 micrograms of α factor to induce cell division arrest. α-MF, alpha mating factor. RA, Arg-1261 to Ala.

    Techniques Used: Transformation Assay, Plasmid Preparation, Activation Assay, Activity Assay

    28) Product Images from "Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation"

    Article Title: Elucidating the Mechanism by which Compensatory Mutations Rescue an HIV-1 Matrix Mutant Defective for Gag Membrane Targeting and Envelope Glycoprotein Incorporation

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2015.01.018

    Release of 29KE/31KE from T-cell lines. VSV-G pseudotyped viruses were generated by transfecting 293T cells with the HIV-1 mutants indicated and a VSV-G expression vector. These viruses were normalized by RT assay and used to infect Jurkat and MT4 T-cell
    Figure Legend Snippet: Release of 29KE/31KE from T-cell lines. VSV-G pseudotyped viruses were generated by transfecting 293T cells with the HIV-1 mutants indicated and a VSV-G expression vector. These viruses were normalized by RT assay and used to infect Jurkat and MT4 T-cell

    Techniques Used: Generated, Expressing, Plasmid Preparation

    29) Product Images from "Curcumin suppresses transforming growth factor-β1-induced cardiac fibroblast differentiation via inhibition of Smad-2 and p38 MAPK signaling pathways"

    Article Title: Curcumin suppresses transforming growth factor-β1-induced cardiac fibroblast differentiation via inhibition of Smad-2 and p38 MAPK signaling pathways

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2016.2969

    Curcumin suppressed α-smooth muscle actin (SMA) and collagen I (ColI) protein expression levels. (A) Western blotting and corresponding densitometric quantification of α-SMA, ColI and internal reference GAPDH expression in cardiac fibroblasts
    Figure Legend Snippet: Curcumin suppressed α-smooth muscle actin (SMA) and collagen I (ColI) protein expression levels. (A) Western blotting and corresponding densitometric quantification of α-SMA, ColI and internal reference GAPDH expression in cardiac fibroblasts

    Techniques Used: Expressing, Western Blot

    Reverse transcription-quantitative polymerase chain reaction analysis of (A) α-smooth muscle actin (SMA) and (B) collagen I (ColI) mRNA expression levlels in cardiac fibroblasts (CFs) treated with or without 5, 10 or 20 µmol/l curcumin
    Figure Legend Snippet: Reverse transcription-quantitative polymerase chain reaction analysis of (A) α-smooth muscle actin (SMA) and (B) collagen I (ColI) mRNA expression levlels in cardiac fibroblasts (CFs) treated with or without 5, 10 or 20 µmol/l curcumin

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Smad2 and p38 inhibitors, SB431542 and SB20380 respectively, suppressed (A) α-smooth muscle actin (SMA) and (B) collagen I (ColI) expression levels. Cardiac fibroblasts (CFs) were treated with 10 ng/ml transforming growth factor (TGF)-β1
    Figure Legend Snippet: Smad2 and p38 inhibitors, SB431542 and SB20380 respectively, suppressed (A) α-smooth muscle actin (SMA) and (B) collagen I (ColI) expression levels. Cardiac fibroblasts (CFs) were treated with 10 ng/ml transforming growth factor (TGF)-β1

    Techniques Used: Expressing

    30) Product Images from "Tau Protects Microtubules in the Axon from Severing by Katanin"

    Article Title: Tau Protects Microtubules in the Axon from Severing by Katanin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5392-05.2006

    Quantitative analysis of MAP levels after treatment with siRNAs. Control siRNA and siRNAs to MAP2, MAP1b, and tau were transfected into hippocampal neurons. Cultures were fixed 1, 3, and 5 d, respectively, after transfection and immunostained for each MAP to evaluate its level as a result of the siRNA. Immunofluorescence indicates depletion of MAP2, MAP1b, and tau in siRNA-treated neurons ( b , d , f ), compared with control siRNA-treated neurons ( B , D , F ), is shown. A , C , E , The quantification of total fluorescence intensity within the soma revealing the progressive loss of MAP2, MAP1b, and tau depletion by siRNA. By day 5, the loss of MAP2, MAP1b, and tau in siRNA treated neurons are 95, 99, and 99%, respectively. G , Western blot of whole-cell extracts probed with the MAP2, MAP1b, and tau Ab, confirming the protein-lowering effect. GAPDH was used as the internal control. Error bars represent SE.
    Figure Legend Snippet: Quantitative analysis of MAP levels after treatment with siRNAs. Control siRNA and siRNAs to MAP2, MAP1b, and tau were transfected into hippocampal neurons. Cultures were fixed 1, 3, and 5 d, respectively, after transfection and immunostained for each MAP to evaluate its level as a result of the siRNA. Immunofluorescence indicates depletion of MAP2, MAP1b, and tau in siRNA-treated neurons ( b , d , f ), compared with control siRNA-treated neurons ( B , D , F ), is shown. A , C , E , The quantification of total fluorescence intensity within the soma revealing the progressive loss of MAP2, MAP1b, and tau depletion by siRNA. By day 5, the loss of MAP2, MAP1b, and tau in siRNA treated neurons are 95, 99, and 99%, respectively. G , Western blot of whole-cell extracts probed with the MAP2, MAP1b, and tau Ab, confirming the protein-lowering effect. GAPDH was used as the internal control. Error bars represent SE.

    Techniques Used: Transfection, Immunofluorescence, Fluorescence, Western Blot

    MAP2c protects microtubules from being severed by P60-katanin, but MAP1b does not. A , C , E , G , GFP–P60-katanin in green and immunostain for either plasmid-expressed MAP2 or MAP1b in red. B , D , F , H , Immunostains for microtubules. A , B , Cells that are not overexpressing P60-katanin. As shown in A and B , MAP1b expression does not cause abnormal bundling of microtubules. As shown in C and D , a cell overexpressing MAP1b and P60-katanin shows only a scattering of very short microtubules and severely reduced microtubule levels. (A neighboring cell not expressing MAP1b or P60-katanin displays a normal microtubule array). As shown in E and F , MAP2c expression causes the formation of dense bundles of microtubules. As shown in G and H , the microtubules in MAP2c-expressing cells show no indication of severing by overexpression of P60-katanin, and the microtubule mass is not reduced. Scale bar: A–H , 30 μm.
    Figure Legend Snippet: MAP2c protects microtubules from being severed by P60-katanin, but MAP1b does not. A , C , E , G , GFP–P60-katanin in green and immunostain for either plasmid-expressed MAP2 or MAP1b in red. B , D , F , H , Immunostains for microtubules. A , B , Cells that are not overexpressing P60-katanin. As shown in A and B , MAP1b expression does not cause abnormal bundling of microtubules. As shown in C and D , a cell overexpressing MAP1b and P60-katanin shows only a scattering of very short microtubules and severely reduced microtubule levels. (A neighboring cell not expressing MAP1b or P60-katanin displays a normal microtubule array). As shown in E and F , MAP2c expression causes the formation of dense bundles of microtubules. As shown in G and H , the microtubules in MAP2c-expressing cells show no indication of severing by overexpression of P60-katanin, and the microtubule mass is not reduced. Scale bar: A–H , 30 μm.

    Techniques Used: Plasmid Preparation, Expressing, Over Expression

    Depletion of tau from cultured hippocampal neurons increases sensitivity to katanin-induced microtubule severing, whereas depletion of MAP1b or MAP2 does not. A–F , Microtubule immunostains of stage III hippocampal neurons. A , A neuron transfected with control siRNA and GFP. B , A neuron transfected with control siRNA and P60-katanin. C , A neuron transfected with MAP1b siRNA and GFP. D , A neuron transfected with MAP1b siRNA and P60-katanin. E , A neuron transfected with tau siRNA and GFP (axon is directed upwards in the panel). F , A neuron transfected with tau siRNA and P60-katanin. Note that the axons show no diminution in fluorescence intensity as a result of overexpression of P60-katanin in either control siRNA or MAP1b siRNA-treated neurons. However, a dramatic diminution was detected in tau-depleted neurons overexpressing P60-katanin. a–f , Glow-scale pseudocolored images of the axons in A–F , with white indicating the highest level, purple indicating the lowest level, and shades of red, orange, and yellow indicating intermediate levels. G , The quantification of microtubule mass in cell bodies, in minor processes, and in axons of stage III neurons in each group. Overexpression of P60-katanin in either control group or the three MAP siRNA-treated groups leads to a 33–40% diminution in microtubule mass from cell bodies ( p
    Figure Legend Snippet: Depletion of tau from cultured hippocampal neurons increases sensitivity to katanin-induced microtubule severing, whereas depletion of MAP1b or MAP2 does not. A–F , Microtubule immunostains of stage III hippocampal neurons. A , A neuron transfected with control siRNA and GFP. B , A neuron transfected with control siRNA and P60-katanin. C , A neuron transfected with MAP1b siRNA and GFP. D , A neuron transfected with MAP1b siRNA and P60-katanin. E , A neuron transfected with tau siRNA and GFP (axon is directed upwards in the panel). F , A neuron transfected with tau siRNA and P60-katanin. Note that the axons show no diminution in fluorescence intensity as a result of overexpression of P60-katanin in either control siRNA or MAP1b siRNA-treated neurons. However, a dramatic diminution was detected in tau-depleted neurons overexpressing P60-katanin. a–f , Glow-scale pseudocolored images of the axons in A–F , with white indicating the highest level, purple indicating the lowest level, and shades of red, orange, and yellow indicating intermediate levels. G , The quantification of microtubule mass in cell bodies, in minor processes, and in axons of stage III neurons in each group. Overexpression of P60-katanin in either control group or the three MAP siRNA-treated groups leads to a 33–40% diminution in microtubule mass from cell bodies ( p

    Techniques Used: Cell Culture, Transfection, Fluorescence, Over Expression

    Effects of depleting MAP2, MAP1b, and tau on the development of hippocampal neurons in culture. Hippocampal neurons were divided into four groups, treated with control siRNA or siRNA to MAP2, MAP1b, or tau, respectively. Replating of neurons was done 2 d after siRNA treatment. Two days after replating, neurons were fixed and stained for microtubules. A , Pie graphs for the ratio of stage I, II, and III hippocampal neurons in each group. There is no significant difference between the control group and the groups depleted of MAP2 or tau. In contrast, depleting MAP1b delayed their development ( p
    Figure Legend Snippet: Effects of depleting MAP2, MAP1b, and tau on the development of hippocampal neurons in culture. Hippocampal neurons were divided into four groups, treated with control siRNA or siRNA to MAP2, MAP1b, or tau, respectively. Replating of neurons was done 2 d after siRNA treatment. Two days after replating, neurons were fixed and stained for microtubules. A , Pie graphs for the ratio of stage I, II, and III hippocampal neurons in each group. There is no significant difference between the control group and the groups depleted of MAP2 or tau. In contrast, depleting MAP1b delayed their development ( p

    Techniques Used: Staining

    31) Product Images from "Genetic Demonstration of a Redundant Role of Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Mitogen-Activated Protein Kinases in Promoting Fibroblast Proliferation ▿"

    Article Title: Genetic Demonstration of a Redundant Role of Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Mitogen-Activated Protein Kinases in Promoting Fibroblast Proliferation ▿

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00131-10

    Disruption of Erk1 gene attenuates the proliferation rate of primary MEFs in a CD-1 background. (A) Relative expression of ERK1 and ERK2 isoforms in CD-1 MEFs. Total lysates from exponentially proliferating CD-1 MEFs ( n = 4) were analyzed by immunoblotting using antibodies specific for phosphorylated (P) ERK1/ERK2, total ERK1/ERK2, and α-tubulin. (B) MEFs derived from Erk1 − / − embryos (ERK1 −/− ) or wild-type control littermates (WT) were made quiescent and then stimulated with 10% NBCS for the indicated times. Total lysates were analyzed by immunoblotting. (C) Kinetics of ERK2 activity. ERK1 −/− or WT MEFs were treated as in panel B. Cell lysates were prepared, and the phosphotransferase activity of endogenous ERK2 was measured by immune complex kinase assay using MBP and [γ- 32 P]ATP as substrates. The top panel shows an autoradiogram and Coomassie staining of MBP. The lower panel is a bar histogram showing the quantification of 32 P incorporation into MBP. (D) Morphology of ERK1 −/− and WT MEFs. (E) ERK1 −/− or WT MEFs were treated as in panel B. The expression and phosphorylation of Mnk1 and p38 were analyzed by immunoblotting. (F) ERK1 −/− or WT MEFs were made quiescent, pretreated with SB203580 (10 μM) or DMSO (0.1%) for 30 min, and then stimulated with 10% NBCS for 15 min. Total lysates were analyzed by immunoblotting using antibodies specific for phosphorylated MNK1, phosphorylated MK2, and α-tubulin. (G) Proliferation rates of P3 MEFs prepared from Erk1 − / − or wild-type littermate embryos were measured by the MTT assay. Values are expressed as fold increase in cell number and correspond to the mean ± standard error of the mean of five independent MEF preparations. The data are representative of three different experiments. *, P
    Figure Legend Snippet: Disruption of Erk1 gene attenuates the proliferation rate of primary MEFs in a CD-1 background. (A) Relative expression of ERK1 and ERK2 isoforms in CD-1 MEFs. Total lysates from exponentially proliferating CD-1 MEFs ( n = 4) were analyzed by immunoblotting using antibodies specific for phosphorylated (P) ERK1/ERK2, total ERK1/ERK2, and α-tubulin. (B) MEFs derived from Erk1 − / − embryos (ERK1 −/− ) or wild-type control littermates (WT) were made quiescent and then stimulated with 10% NBCS for the indicated times. Total lysates were analyzed by immunoblotting. (C) Kinetics of ERK2 activity. ERK1 −/− or WT MEFs were treated as in panel B. Cell lysates were prepared, and the phosphotransferase activity of endogenous ERK2 was measured by immune complex kinase assay using MBP and [γ- 32 P]ATP as substrates. The top panel shows an autoradiogram and Coomassie staining of MBP. The lower panel is a bar histogram showing the quantification of 32 P incorporation into MBP. (D) Morphology of ERK1 −/− and WT MEFs. (E) ERK1 −/− or WT MEFs were treated as in panel B. The expression and phosphorylation of Mnk1 and p38 were analyzed by immunoblotting. (F) ERK1 −/− or WT MEFs were made quiescent, pretreated with SB203580 (10 μM) or DMSO (0.1%) for 30 min, and then stimulated with 10% NBCS for 15 min. Total lysates were analyzed by immunoblotting using antibodies specific for phosphorylated MNK1, phosphorylated MK2, and α-tubulin. (G) Proliferation rates of P3 MEFs prepared from Erk1 − / − or wild-type littermate embryos were measured by the MTT assay. Values are expressed as fold increase in cell number and correspond to the mean ± standard error of the mean of five independent MEF preparations. The data are representative of three different experiments. *, P

    Techniques Used: Expressing, Derivative Assay, Activity Assay, Immune Complex Kinase Assay, Staining, MTT Assay

    32) Product Images from "Multiple Targeting Modules on Peroxisomal Proteins Are Not Redundant: Discrete Functions of Targeting Signals within Pmp47 and Pex8p"

    Article Title: Multiple Targeting Modules on Peroxisomal Proteins Are Not Redundant: Discrete Functions of Targeting Signals within Pmp47 and Pex8p

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E03-11-0810

    PTS1 of Pex8p increases targeting efficiency to basal peroxisomes. Strains in which the chromosomal copy of PEX8 or PEX14 was replaced with one containing sequences for HA or HA-SKL (as indicated) were cultured in oleate medium or YPD and then subjected to organelle fractionation. (A) Immunoblots using anti-HA, or G6PDH, against an equal percentage of fractions are shown. The distribution of glucose-6-phosphate dehydrogenase among fractions was similar in all strains; only one is shown. Asterisks denote a comigrating band present in equal intensity in the 55K supernatants of all strains, such that the contribution of Pex8p-HA proteins to this band is minimal. (B) Percentage of Pex8-HA or Pex8-HA-SKL in the 13.5K peroxisomal fraction (compared with the sum in 13.5 and 55K pellets) is shown, based on densitometry. The data represent five separate experiments with means + SE shown. In every experiment, the addition of SKL increased targeting efficiency.
    Figure Legend Snippet: PTS1 of Pex8p increases targeting efficiency to basal peroxisomes. Strains in which the chromosomal copy of PEX8 or PEX14 was replaced with one containing sequences for HA or HA-SKL (as indicated) were cultured in oleate medium or YPD and then subjected to organelle fractionation. (A) Immunoblots using anti-HA, or G6PDH, against an equal percentage of fractions are shown. The distribution of glucose-6-phosphate dehydrogenase among fractions was similar in all strains; only one is shown. Asterisks denote a comigrating band present in equal intensity in the 55K supernatants of all strains, such that the contribution of Pex8p-HA proteins to this band is minimal. (B) Percentage of Pex8-HA or Pex8-HA-SKL in the 13.5K peroxisomal fraction (compared with the sum in 13.5 and 55K pellets) is shown, based on densitometry. The data represent five separate experiments with means + SE shown. In every experiment, the addition of SKL increased targeting efficiency.

    Techniques Used: Cell Culture, Fractionation, Western Blot

    33) Product Images from "A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis"

    Article Title: A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2016.102

    RIP3 and MLKL are required for Bak activation and mitochondrial perturbations during BV6/Dexa-induced necroptosis. Tanoue cells were transiently transfected with two distinct siRNAs targeting RIP3, MLKL or control siRNA, and treated for 6 h with 3 μ M BV6 and 200 μ M Dexa. MEFs were treated for 12 h with 5 μ M BV6 and 200 μ M Dexa in the presence of 20 μ M zVAD.fmk. ( a and b ) Bak ( a ) or Bax ( b ) activation was determined by immunoprecipitation using active conformation-specific antibodies. Protein expression of Bak, Bax, RIP3 and MLKL were analyzed by western blotting. β -Actin served as loading control. ( c and d ) Loss of MMP was determined by TMRM staining and flow cytometry ( c ) or ImageXpress Micro XLS system ( d ). Mean and S.D. of three independent experiments performed in triplicate are shown; * P
    Figure Legend Snippet: RIP3 and MLKL are required for Bak activation and mitochondrial perturbations during BV6/Dexa-induced necroptosis. Tanoue cells were transiently transfected with two distinct siRNAs targeting RIP3, MLKL or control siRNA, and treated for 6 h with 3 μ M BV6 and 200 μ M Dexa. MEFs were treated for 12 h with 5 μ M BV6 and 200 μ M Dexa in the presence of 20 μ M zVAD.fmk. ( a and b ) Bak ( a ) or Bax ( b ) activation was determined by immunoprecipitation using active conformation-specific antibodies. Protein expression of Bak, Bax, RIP3 and MLKL were analyzed by western blotting. β -Actin served as loading control. ( c and d ) Loss of MMP was determined by TMRM staining and flow cytometry ( c ) or ImageXpress Micro XLS system ( d ). Mean and S.D. of three independent experiments performed in triplicate are shown; * P

    Techniques Used: Activation Assay, Transfection, Immunoprecipitation, Expressing, Western Blot, Staining, Flow Cytometry, Cytometry

    34) Product Images from "Relative importance of βcyto- and γcyto-actin in primary mouse embryonic fibroblasts"

    Article Title: Relative importance of βcyto- and γcyto-actin in primary mouse embryonic fibroblasts

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-07-0503

    Caldesmon smooth muscle isoform protein expression was up-regulated in β cyto -actin–deficient MEFs. (A) Representative Western blot analysis of CT and KO MEFs blotted with caldesmon (Cald1 sm ), CNN1, Sm22α, MLC2, and pMLC2; GAPDH served as loading control. (B–D) Relative protein expression normalized to GAPDH and relative to the paired embryo control. * p
    Figure Legend Snippet: Caldesmon smooth muscle isoform protein expression was up-regulated in β cyto -actin–deficient MEFs. (A) Representative Western blot analysis of CT and KO MEFs blotted with caldesmon (Cald1 sm ), CNN1, Sm22α, MLC2, and pMLC2; GAPDH served as loading control. (B–D) Relative protein expression normalized to GAPDH and relative to the paired embryo control. * p

    Techniques Used: Expressing, Western Blot

    35) Product Images from "Digoxin inhibits PDGF-BB-induced VSMC proliferation and migration through an increase in ILK signaling and attenuates neointima formation following carotid injury"

    Article Title: Digoxin inhibits PDGF-BB-induced VSMC proliferation and migration through an increase in ILK signaling and attenuates neointima formation following carotid injury

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2015.2320

    Effect of digoxin on the regulation of smooth muscle cell contractile profiles. Vascular smooth muscle cells (VSMCs) were pre-cultured in the serum-free medium for 24 h and then treated with the digoxin (100 nM) for 24 h and then stimulated with platelet-derived growth factor (PDGF)-BB (25 µ g/l) for 24 h. (A) Protein levels of SM22a, calponin and SM α-actin were determined by western blot analysis and quantified by densitometry. β-actin was used as an internal control. (B) Bar graphs showing the quantification of the western blots; results are expressed as percentages of the control. The results are expressed as relative values from 3 independent experiments. ( # P
    Figure Legend Snippet: Effect of digoxin on the regulation of smooth muscle cell contractile profiles. Vascular smooth muscle cells (VSMCs) were pre-cultured in the serum-free medium for 24 h and then treated with the digoxin (100 nM) for 24 h and then stimulated with platelet-derived growth factor (PDGF)-BB (25 µ g/l) for 24 h. (A) Protein levels of SM22a, calponin and SM α-actin were determined by western blot analysis and quantified by densitometry. β-actin was used as an internal control. (B) Bar graphs showing the quantification of the western blots; results are expressed as percentages of the control. The results are expressed as relative values from 3 independent experiments. ( # P

    Techniques Used: Cell Culture, Derivative Assay, Western Blot

    36) Product Images from "Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit"

    Article Title: Polarized Axonal Surface Expression of Neuronal KCNQ Potassium Channels Is Regulated by Calmodulin Interaction with KCNQ2 Subunit

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103655

    The R353G mutation reduces CD4-Q2C expression in the axon. (A) Permeabilized immunostaining of wild-type (WT) or R353G mutant CD4-Q2C in hippocampal neurons (DIV 7–8) cotransfected with GFP. Neuronal soma and dendrites were identified by MAP2 immunostaining (middle). The insets in the representative inverted images of total CD4-Q2C show the initial segment (AIS) and distal segment (A) of the MAP2-negative axon in transfected neurons. Camera lucida drawings (lower) of the neuronal images (upper) show the soma and dendrites (gray) and an axon (black). The R353G mutation reduced but did not abolish total (surface and intracellular) expression of CD4-Q2C from the axon. Scale bars are 20 µm. (B) Background subtracted, mean intensity of total CD4 fluorescence in the AIS, distal axons, soma, and major dendrites. AU, arbitrary unit. The sample number for each construct used was as follows: WT (n = 23), R353G (n = 18), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (**p
    Figure Legend Snippet: The R353G mutation reduces CD4-Q2C expression in the axon. (A) Permeabilized immunostaining of wild-type (WT) or R353G mutant CD4-Q2C in hippocampal neurons (DIV 7–8) cotransfected with GFP. Neuronal soma and dendrites were identified by MAP2 immunostaining (middle). The insets in the representative inverted images of total CD4-Q2C show the initial segment (AIS) and distal segment (A) of the MAP2-negative axon in transfected neurons. Camera lucida drawings (lower) of the neuronal images (upper) show the soma and dendrites (gray) and an axon (black). The R353G mutation reduced but did not abolish total (surface and intracellular) expression of CD4-Q2C from the axon. Scale bars are 20 µm. (B) Background subtracted, mean intensity of total CD4 fluorescence in the AIS, distal axons, soma, and major dendrites. AU, arbitrary unit. The sample number for each construct used was as follows: WT (n = 23), R353G (n = 18), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (**p

    Techniques Used: Mutagenesis, Expressing, Immunostaining, Transfection, Fluorescence, Construct

    The A343D mutation abolishes axonal expression of HA-KCNQ3/KCNQ2 channels. (A) Permeabilized immunostaining was performed to visualize total (surface and intracellular) expression of HA-KCNQ3/KCNQ2 WT or mutant (A343D and R353G) channels (inverted images, upper). The axon was identified by the AIS marker phospho IκBα Ser32 (14D4) whereas neuronal soma and dendrites were visualized by MAP2 immunostaining (lower). The A343D but not the R353G mutation abolished total expression of HA-KCNQ3/KCNQ2 channels at the axon. Arrows indicate the AIS. Scale bars are 20 µm. (B) Background subtracted, mean intensity of total HA fluorescence in the AIS, distal axons, soma, and major dendrites. The sample number for each construct was as follows: WT (n = 27), A343D (n = 22), R353G (n = 21), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p
    Figure Legend Snippet: The A343D mutation abolishes axonal expression of HA-KCNQ3/KCNQ2 channels. (A) Permeabilized immunostaining was performed to visualize total (surface and intracellular) expression of HA-KCNQ3/KCNQ2 WT or mutant (A343D and R353G) channels (inverted images, upper). The axon was identified by the AIS marker phospho IκBα Ser32 (14D4) whereas neuronal soma and dendrites were visualized by MAP2 immunostaining (lower). The A343D but not the R353G mutation abolished total expression of HA-KCNQ3/KCNQ2 channels at the axon. Arrows indicate the AIS. Scale bars are 20 µm. (B) Background subtracted, mean intensity of total HA fluorescence in the AIS, distal axons, soma, and major dendrites. The sample number for each construct was as follows: WT (n = 27), A343D (n = 22), R353G (n = 21), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p

    Techniques Used: Mutagenesis, Expressing, Immunostaining, Marker, Fluorescence, Construct

    The A343D mutation impairs HA-KCNQ3/KCNQ2 trafficking from the ER to the axon. (A–B) Pulse-chase assay of wild-type (WT) or mutant (A343D and R353G) HA-KCNQ3/KCNQ2 channels after BFA washout. Permeabilized immunostaining was performed to visualize total (surface and intracellular) expression of HA-KCNQ3/KCNQ2 channels in the soma and AIS (A) as well as in the distal axons and dendrites (B) at indicated time points post-BFA removal (inverted images). The axon was identified by the AIS marker phospho IκBα Ser32 (14D4), whereas neuronal soma and dendrites were visualized by MAP2 immunostaining. Arrows indicate the AIS. Scale bars: 10 µm. (C) Background subtracted, mean intensity of total HA fluorescence in the AIS, distal axon, and major dendrites. Upon BFA removal, the A343D mutation but not the R353G mutation markedly reduced the appearance of HA-KCNQ3/KCNQ2 at the AIS and distal axon for the duration of the 8 hr BFA washout. The sample numbers were (n = 8–22) per time point for each construct. AU, arbitrary unit. Ave ± SEM (*p
    Figure Legend Snippet: The A343D mutation impairs HA-KCNQ3/KCNQ2 trafficking from the ER to the axon. (A–B) Pulse-chase assay of wild-type (WT) or mutant (A343D and R353G) HA-KCNQ3/KCNQ2 channels after BFA washout. Permeabilized immunostaining was performed to visualize total (surface and intracellular) expression of HA-KCNQ3/KCNQ2 channels in the soma and AIS (A) as well as in the distal axons and dendrites (B) at indicated time points post-BFA removal (inverted images). The axon was identified by the AIS marker phospho IκBα Ser32 (14D4), whereas neuronal soma and dendrites were visualized by MAP2 immunostaining. Arrows indicate the AIS. Scale bars: 10 µm. (C) Background subtracted, mean intensity of total HA fluorescence in the AIS, distal axon, and major dendrites. Upon BFA removal, the A343D mutation but not the R353G mutation markedly reduced the appearance of HA-KCNQ3/KCNQ2 at the AIS and distal axon for the duration of the 8 hr BFA washout. The sample numbers were (n = 8–22) per time point for each construct. AU, arbitrary unit. Ave ± SEM (*p

    Techniques Used: Mutagenesis, Pulse Chase, Immunostaining, Expressing, Marker, Fluorescence, Construct

    The HA-KCNQ3/KCNQ2-A343D mutant channels are absent from the ER-negative axon. (A) Permeabilized immunostaining of HA-KCNQ3/KCNQ2 channels (inverted images, left) and MAP2 (middle) were performed in cultured hippocampal neurons (DIV 7) cotransfected with the CD4 proteins harboring the ER retention/retrieval motif (CD4-KDEL, green). The wild type (WT) and R353G mutant channels were found at the axons where the A343D mutant channels and CD4-KDEL proteins were absent. The insets show the major axon. (B) The cultured hippocampal neurons (DIV 5) were treated with vehicle control (No BFA) or BFA (0.75 µg/ml) at 30 min post transfection with HA-KCNQ3 and KCNQ2 wild-type (WT), or mutant (A343D and R353G). At 16 hr post-BFA treatment, permeabilized immunostaining was performed to visualize total (surface and intracellular) expression of HA-KCNQ3/KCNQ2 channels. The axon was identified by the AIS marker phospho IκBα Ser32 (14D4), whereas neuronal soma and dendrites were visualized by MAP2 immunostaining. BFA treatment caused newly synthesized wild-type and all mutant channels to accumulate at perinuclear regions in the soma and dendrites but not axons. Arrows mark the AIS. Scale bars are 20 µm.
    Figure Legend Snippet: The HA-KCNQ3/KCNQ2-A343D mutant channels are absent from the ER-negative axon. (A) Permeabilized immunostaining of HA-KCNQ3/KCNQ2 channels (inverted images, left) and MAP2 (middle) were performed in cultured hippocampal neurons (DIV 7) cotransfected with the CD4 proteins harboring the ER retention/retrieval motif (CD4-KDEL, green). The wild type (WT) and R353G mutant channels were found at the axons where the A343D mutant channels and CD4-KDEL proteins were absent. The insets show the major axon. (B) The cultured hippocampal neurons (DIV 5) were treated with vehicle control (No BFA) or BFA (0.75 µg/ml) at 30 min post transfection with HA-KCNQ3 and KCNQ2 wild-type (WT), or mutant (A343D and R353G). At 16 hr post-BFA treatment, permeabilized immunostaining was performed to visualize total (surface and intracellular) expression of HA-KCNQ3/KCNQ2 channels. The axon was identified by the AIS marker phospho IκBα Ser32 (14D4), whereas neuronal soma and dendrites were visualized by MAP2 immunostaining. BFA treatment caused newly synthesized wild-type and all mutant channels to accumulate at perinuclear regions in the soma and dendrites but not axons. Arrows mark the AIS. Scale bars are 20 µm.

    Techniques Used: Mutagenesis, Immunostaining, Cell Culture, Transfection, Expressing, Marker, Synthesized

    Enrichment of HA-KCNQ3/KCNQ2 channels and CD4-Q2C at the axonal surface. Schematic drawings (not to scale) of a human KCNQ2 subunit (accession #Y15065), including the subunit interaction domain (Sid, amino acids 580–623) [52] , [53] , and CaM-binding domain (amino acids 323–579) [22] , [23] . (B) Immunoblot analysis of CaM in cultured rat hippocampal neurons at 5–9 days in vitro (DIV). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. (C) Schematic drawings (not to scale) of a human CD4 protein, and CD4 fused to KCNQ2 C-terminal tail (CD4-Q2C, [18] ). (D) Surface immunostaining of hippocampal neurons (DIV 7) transfected with KCNQ2 and KCNQ3 containing an extracellular hemagglutinin (HA) epitope (HA-KCNQ3). Neuronal soma and dendrites were visualized by immunostaining for MAP2. Surface HA-KCNQ3/KCNQ2 channels are enriched on a MAP2-negative neurite that originates directly from the soma. (E) Pseudo-color image of the inset in Fig. 1D displays differences in the surface HA intensity. Surface HA-KCNQ3/KCNQ2 channels are enriched at the initial segment of an axon. (F) Surface immunostaining of hippocampal neurons (DIV 8) transfected with CD4, or CD4-Q2C. (G) Pseudo-color images of the insets in Fig. 1F display differences in the surface CD4 intensity. Fusion of KCNQ2 C-terminal tail enriches CD4 at the axonal surface. Camera lucida drawings of the neuronal images (D, F) show the soma and dendrites (gray) and an axon (black). Arrows mark the main axon. Scale bars are 20 µm.
    Figure Legend Snippet: Enrichment of HA-KCNQ3/KCNQ2 channels and CD4-Q2C at the axonal surface. Schematic drawings (not to scale) of a human KCNQ2 subunit (accession #Y15065), including the subunit interaction domain (Sid, amino acids 580–623) [52] , [53] , and CaM-binding domain (amino acids 323–579) [22] , [23] . (B) Immunoblot analysis of CaM in cultured rat hippocampal neurons at 5–9 days in vitro (DIV). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. (C) Schematic drawings (not to scale) of a human CD4 protein, and CD4 fused to KCNQ2 C-terminal tail (CD4-Q2C, [18] ). (D) Surface immunostaining of hippocampal neurons (DIV 7) transfected with KCNQ2 and KCNQ3 containing an extracellular hemagglutinin (HA) epitope (HA-KCNQ3). Neuronal soma and dendrites were visualized by immunostaining for MAP2. Surface HA-KCNQ3/KCNQ2 channels are enriched on a MAP2-negative neurite that originates directly from the soma. (E) Pseudo-color image of the inset in Fig. 1D displays differences in the surface HA intensity. Surface HA-KCNQ3/KCNQ2 channels are enriched at the initial segment of an axon. (F) Surface immunostaining of hippocampal neurons (DIV 8) transfected with CD4, or CD4-Q2C. (G) Pseudo-color images of the insets in Fig. 1F display differences in the surface CD4 intensity. Fusion of KCNQ2 C-terminal tail enriches CD4 at the axonal surface. Camera lucida drawings of the neuronal images (D, F) show the soma and dendrites (gray) and an axon (black). Arrows mark the main axon. Scale bars are 20 µm.

    Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Cell Culture, In Vitro, Immunostaining, Transfection

    CD4-Q2C accumulates in the ER after BFA treatment. (A) Schematic drawings of a hippocampal neuron containing a continuous network of the ER from the soma to the dendrites. Treatment with brefeldin-A (BFA) leads to inhibition of anterograde transport from the ER to the Golgi complex [44] . (B–C) The cultured hippocampal neurons (DIV 5) were treated with vehicle control (No BFA) or BFA (0.75 µg/ml) at 30 min post transfection with GFP and CD4-Q2C wild-type (WT) (B), I340E mutant (C), or R353G mutant (D). At 16 hr post-BFA treatment, permeabilized immunostaining was performed to visualize total (surface and intracellular) expression of CD4-Q2C (inverted images, upper). BFA treatment caused newly synthesized wild-type and all mutant proteins to accumulate at perinuclear regions in the soma and proximal dendrites. Arrows mark the main axon identified by the lack of MAP2 immunostaining in the neurons cotransfected with GFP. Camera lucida drawings (lower) show the soma and dendrites (gray) and an axon (black). Scale bars are 20 µm.
    Figure Legend Snippet: CD4-Q2C accumulates in the ER after BFA treatment. (A) Schematic drawings of a hippocampal neuron containing a continuous network of the ER from the soma to the dendrites. Treatment with brefeldin-A (BFA) leads to inhibition of anterograde transport from the ER to the Golgi complex [44] . (B–C) The cultured hippocampal neurons (DIV 5) were treated with vehicle control (No BFA) or BFA (0.75 µg/ml) at 30 min post transfection with GFP and CD4-Q2C wild-type (WT) (B), I340E mutant (C), or R353G mutant (D). At 16 hr post-BFA treatment, permeabilized immunostaining was performed to visualize total (surface and intracellular) expression of CD4-Q2C (inverted images, upper). BFA treatment caused newly synthesized wild-type and all mutant proteins to accumulate at perinuclear regions in the soma and proximal dendrites. Arrows mark the main axon identified by the lack of MAP2 immunostaining in the neurons cotransfected with GFP. Camera lucida drawings (lower) show the soma and dendrites (gray) and an axon (black). Scale bars are 20 µm.

    Techniques Used: Inhibition, Cell Culture, Transfection, Mutagenesis, Immunostaining, Expressing, Synthesized

    CaM1234 reduces enrichment of HA-KCNQ3/KCNQ2 channels at the axonal surface. (A) Surface expression of HA-KCNQ3/KCNQ2 channels in hippocampal neurons (DIV 7–8) cotransfected with empty vector (pcDNA3), CaM wild-type (WT) or Ca 2+ -insensitive mutant CaM (CaM1234). Endogenous and transfected CaM proteins were immunostained with anti-CaM antibodies (CaM, lower inverted images). HA-KCNQ3/KCNQ2 channels were enriched at the axonal surface in the presence of CaM WT or CaM1234. Arrows mark the main axon. Scale bars are 20 µm. (B) Overexpression of CaM WT or CaM1234 did not grossly affect neuronal polarity as indicated by immunostaining of the somatodendritic marker MAP2 and the AIS marker ankryin-G. Arrows indicate the AIS. Scale bars are 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by 33% by coexpression with CaM1234 (n = 21) compared to coexpression with CaM WT (n = 33) or empty vector pcDNA3 (n = 43). The surface “AIS/Axon” ratio of CD4-Q2C was increased by coexpression with CaM1234 compared to CaM WT but not pcDNA3. (D) Background subtracted, mean intensity of the CaM fluorescence in the soma of transfected and untransfected neurons (n = 23). (E) Background subtracted, mean intensity of surface and total (surface and intracellular) HA fluorescence in transfected and untransfected neurons. CaM1234 modestly decreased surface expression of HA-KCNQ3/KCNQ2 channels at the AIS and axon. (D, E) AU, arbitrary unit. Ave ± SEM (*p
    Figure Legend Snippet: CaM1234 reduces enrichment of HA-KCNQ3/KCNQ2 channels at the axonal surface. (A) Surface expression of HA-KCNQ3/KCNQ2 channels in hippocampal neurons (DIV 7–8) cotransfected with empty vector (pcDNA3), CaM wild-type (WT) or Ca 2+ -insensitive mutant CaM (CaM1234). Endogenous and transfected CaM proteins were immunostained with anti-CaM antibodies (CaM, lower inverted images). HA-KCNQ3/KCNQ2 channels were enriched at the axonal surface in the presence of CaM WT or CaM1234. Arrows mark the main axon. Scale bars are 20 µm. (B) Overexpression of CaM WT or CaM1234 did not grossly affect neuronal polarity as indicated by immunostaining of the somatodendritic marker MAP2 and the AIS marker ankryin-G. Arrows indicate the AIS. Scale bars are 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by 33% by coexpression with CaM1234 (n = 21) compared to coexpression with CaM WT (n = 33) or empty vector pcDNA3 (n = 43). The surface “AIS/Axon” ratio of CD4-Q2C was increased by coexpression with CaM1234 compared to CaM WT but not pcDNA3. (D) Background subtracted, mean intensity of the CaM fluorescence in the soma of transfected and untransfected neurons (n = 23). (E) Background subtracted, mean intensity of surface and total (surface and intracellular) HA fluorescence in transfected and untransfected neurons. CaM1234 modestly decreased surface expression of HA-KCNQ3/KCNQ2 channels at the AIS and axon. (D, E) AU, arbitrary unit. Ave ± SEM (*p

    Techniques Used: Expressing, Plasmid Preparation, Chick Chorioallantoic Membrane Assay, Mutagenesis, Transfection, Over Expression, Immunostaining, Marker, Fluorescence

    The BFNC R353G mutation blocks axonal enrichment of surface CD4-Q2C. (A) The R353G mutation reduced but did not abolish co-immunoprecipitation of CaM with CD4-Q2C from transfected HEK293T cells. β-actin served as a loading control for total cell lysates (B) Surface immunostaining of WT or R353G mutant CD4-Q2C in hippocampal neurons (DIV 7–8). Camera lucida drawings (lower) of the inverted images of surface CD4-Q2C (upper) show the soma and dendrites (gray) and an axon (black). The axon was identified by the lack of MAP2 immunostaining in the neurons cotransfected with GFP. The R353G mutation blocked enrichment of CD4-Q2C on the axonal surface by increasing its somatodendritic surface expression. Arrows mark the main axon. Scale bars are 20 µm. (C) In comparison to WT, the surface “Axon/Dendrite” fluorescence ratio of CD4-Q2C was reduced to 1 by the R353G mutation, whereas the surface “AIS/Axon” ratio was unaffected. (D) Background subtracted, mean intensity of surface CD4 fluorescence in the AIS, distal axons, soma, and major dendrites. The R353G mutation increased CD4-Q2C expression at the somatodendritic surface compared to WT. The sample number for each construct used in (C, D) was as follows: WT (n = 23), R353G (n = 18), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p
    Figure Legend Snippet: The BFNC R353G mutation blocks axonal enrichment of surface CD4-Q2C. (A) The R353G mutation reduced but did not abolish co-immunoprecipitation of CaM with CD4-Q2C from transfected HEK293T cells. β-actin served as a loading control for total cell lysates (B) Surface immunostaining of WT or R353G mutant CD4-Q2C in hippocampal neurons (DIV 7–8). Camera lucida drawings (lower) of the inverted images of surface CD4-Q2C (upper) show the soma and dendrites (gray) and an axon (black). The axon was identified by the lack of MAP2 immunostaining in the neurons cotransfected with GFP. The R353G mutation blocked enrichment of CD4-Q2C on the axonal surface by increasing its somatodendritic surface expression. Arrows mark the main axon. Scale bars are 20 µm. (C) In comparison to WT, the surface “Axon/Dendrite” fluorescence ratio of CD4-Q2C was reduced to 1 by the R353G mutation, whereas the surface “AIS/Axon” ratio was unaffected. (D) Background subtracted, mean intensity of surface CD4 fluorescence in the AIS, distal axons, soma, and major dendrites. The R353G mutation increased CD4-Q2C expression at the somatodendritic surface compared to WT. The sample number for each construct used in (C, D) was as follows: WT (n = 23), R353G (n = 18), and untransfected (n = 15). AU, arbitrary unit. Ave ± SEM (*p

    Techniques Used: Mutagenesis, Immunoprecipitation, Chick Chorioallantoic Membrane Assay, Transfection, Immunostaining, Expressing, Fluorescence, Construct

    37) Product Images from "Effects of high glucose on integrin activity and fibronectin matrix assembly by mesangial cells"

    Article Title: Effects of high glucose on integrin activity and fibronectin matrix assembly by mesangial cells

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-03-0800

    Mn 2+ stimulates assembly of FN matrix in normal glucose. Mesangial cells were grown in normal or high glucose media and then labeled with an anti–α5β1 integrin antibody for analysis by flow cytometry (A), replated on FN-coated coverslips (B), or fixed and stained with IC3 anti-rat FN monoclonal antibody (C). (A) The graph is representative of two independent experiments. (B) Cells were incubated for 1 h at 37°C with or without 1.0 mM MnCl 2 before unattached cells were washed away. Attached cells were fixed and stained with rhodamine–phalloidin, and 15 fields/condition were counted. Images are representative; scale bar, 100 μm. Bar graph shows the mean of two to four independent experiments ± 1 SD; * p
    Figure Legend Snippet: Mn 2+ stimulates assembly of FN matrix in normal glucose. Mesangial cells were grown in normal or high glucose media and then labeled with an anti–α5β1 integrin antibody for analysis by flow cytometry (A), replated on FN-coated coverslips (B), or fixed and stained with IC3 anti-rat FN monoclonal antibody (C). (A) The graph is representative of two independent experiments. (B) Cells were incubated for 1 h at 37°C with or without 1.0 mM MnCl 2 before unattached cells were washed away. Attached cells were fixed and stained with rhodamine–phalloidin, and 15 fields/condition were counted. Images are representative; scale bar, 100 μm. Bar graph shows the mean of two to four independent experiments ± 1 SD; * p

    Techniques Used: Labeling, Flow Cytometry, Cytometry, Staining, Incubation

    38) Product Images from "Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection"

    Article Title: Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection

    Journal: Gene Therapy

    doi: 10.1038/gt.2010.133

    In vivo mRNA and protein down-modulation. ( a , b ) Nude mice-bearing 15PC3 tumors were injected with indicated amount of EZN-3920 or EZN-3046 control (iv, qd × 5). Tumors were harvested and analyzed for HER3 mRNA on day 6. mRNA down-modulation of HER3 or HIF-1α in 15PC3 tumors were determined by RT-quantitative PCR (qPCR) with TaqMan gene expression assays. Data are means±s.e. ( n =8) ( c ) Nude mice-bearing 15PC3 tumors were injected with indicated amount of EZN-3920 (iv, q3d × 6). Two days after the last dosing, the tumor tissues were harvested. Protein levels of HER3, p-AKT, AKT, mitogen-activated protein kinase (MAPK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in tumor samples were analyzed with western blot analysis. Data from individual animals are shown. Relative quantitation of pAkt and Akt levels (indicated at the bottom) were carried out by using Multi Guage Image Analysis software (Fujifilm Life Science, Minato-ku, Tokyo, Japan). ( d ) Nude mice-bearing 15PC3 tumors were injected with indicated amount of EZN-3920 (iv, q3d × 6). Two days after the last dosing, the liver tissues were harvested. The top panel shows the relative mRNA level of HER3 determined by RT-qPCR. Protein levels of HER3 and tubulin were analyzed with western blot analysis (lower panel). Data from individual animals are shown. Quantitation of relative HER3 levels was carried out by using the Multi Guage Image Analysis software. The numbers denote the % of normalized ratios of HER3 over tubulin.
    Figure Legend Snippet: In vivo mRNA and protein down-modulation. ( a , b ) Nude mice-bearing 15PC3 tumors were injected with indicated amount of EZN-3920 or EZN-3046 control (iv, qd × 5). Tumors were harvested and analyzed for HER3 mRNA on day 6. mRNA down-modulation of HER3 or HIF-1α in 15PC3 tumors were determined by RT-quantitative PCR (qPCR) with TaqMan gene expression assays. Data are means±s.e. ( n =8) ( c ) Nude mice-bearing 15PC3 tumors were injected with indicated amount of EZN-3920 (iv, q3d × 6). Two days after the last dosing, the tumor tissues were harvested. Protein levels of HER3, p-AKT, AKT, mitogen-activated protein kinase (MAPK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in tumor samples were analyzed with western blot analysis. Data from individual animals are shown. Relative quantitation of pAkt and Akt levels (indicated at the bottom) were carried out by using Multi Guage Image Analysis software (Fujifilm Life Science, Minato-ku, Tokyo, Japan). ( d ) Nude mice-bearing 15PC3 tumors were injected with indicated amount of EZN-3920 (iv, q3d × 6). Two days after the last dosing, the liver tissues were harvested. The top panel shows the relative mRNA level of HER3 determined by RT-qPCR. Protein levels of HER3 and tubulin were analyzed with western blot analysis (lower panel). Data from individual animals are shown. Quantitation of relative HER3 levels was carried out by using the Multi Guage Image Analysis software. The numbers denote the % of normalized ratios of HER3 over tubulin.

    Techniques Used: In Vivo, Mouse Assay, Injection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Quantitation Assay, Software, Quantitative RT-PCR

    39) Product Images from "Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis"

    Article Title: Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aaj1789

    SIRT1 activation modulates the lung myeloid landscape in Mtb -infected mice
    Figure Legend Snippet: SIRT1 activation modulates the lung myeloid landscape in Mtb -infected mice

    Techniques Used: Activation Assay, Infection, Mouse Assay

    Mtb Infection down-regulates SIRT1 expression
    Figure Legend Snippet: Mtb Infection down-regulates SIRT1 expression

    Techniques Used: Infection, Expressing

    SIRT1 activators enhance control of Mtb growth
    Figure Legend Snippet: SIRT1 activators enhance control of Mtb growth

    Techniques Used:

    SIRT1 activators normalize Mtb -induced inflammatory responses
    Figure Legend Snippet: SIRT1 activators normalize Mtb -induced inflammatory responses

    Techniques Used:

    SIRT1 activation reduces Mtb growth in mice
    Figure Legend Snippet: SIRT1 activation reduces Mtb growth in mice

    Techniques Used: Activation Assay, Mouse Assay

    SIRT1 activation restricts Mtb growth in mice
    Figure Legend Snippet: SIRT1 activation restricts Mtb growth in mice

    Techniques Used: Activation Assay, Mouse Assay

    SIRT1 activation reduces TB-associated tissue pathology and inflammation in mice
    Figure Legend Snippet: SIRT1 activation reduces TB-associated tissue pathology and inflammation in mice

    Techniques Used: Activation Assay, Mouse Assay

    40) Product Images from "Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket"

    Article Title: Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket

    Journal: eLife

    doi: 10.7554/eLife.18492

    Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004
    Figure Legend Snippet: Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004

    Techniques Used: Activation Assay, Staining, Western Blot, Mouse Assay, Expressing

    41) Product Images from "Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket"

    Article Title: Spontaneous activation of visual pigments in relation to openness/closedness of chromophore-binding pocket

    Journal: eLife

    doi: 10.7554/eLife.18492

    Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004
    Figure Legend Snippet: Measurement of spontaneous-activation rate of E122Q-rhodopsin. ( A ) Paraffin sections of 2.5-month-old Rho WT/WT ;Gcaps -/- (left) and Rho E122Q/E122Q ;Gcaps -/- (right) retinas stained by haematoxylin and eosin showing normal rod morphology. Similar results were found in altogether 3 sets of experiments. ( B ) Western blots from retinal extracts of Rho WT/WT ;Gcaps -/- (different animal in each of the left two columns) and Rho E122Q/E122Q ;Gcaps -/- mice (different animal in each of right two columns) showing normal expression of various phototransduction protein components. RHO: rhodopsin; G tα : α subunit of transducin; PDE6: phosphodiesterase isoform 6; CNGA1: A1 subunit of cyclic nucleotide-gated (CNG) channel; CNGB1: B1 subunit of CNG channel; ARR1: Arrestin 1; RGS9: regulator of G protein signaling isoform 9; GAPDH: glyceraldehyde 3-phosphate dehydrogenase (control for protein amount). ( C ) Sample 10 min recordings from a Rho WT/WT ;Gcaps -/- rod (left) and a Rho E122Q/E122Q ;Gcaps -/- rod (right) in darkness. Traces (continuous from top to bottom) were low-pass filtered at 3 Hz. Quantal events were identified based on amplitude and kinetics (see Text) and are marked by asterisks. ( D ) Poisson analysis of dark recordings collected from all Rho E122Q/E122Q ;Gcaps -/- rods. Bars indicate the measured probabilities of observing 0, 1, 2 and 3 events in 100 s epochs. A total of 118 epochs were analyzed. Red lines give the fit by the Poisson distribution with a mean event rate of 0.0023 s −1 cell −1 . ( E ) Difference power spectrum (square symbols) of a Rho E122Q/E122Q ;Gcaps -/- rod fitted with the power spectrum (curve) of the single-photon-response function. DOI: http://dx.doi.org/10.7554/eLife.18492.003 10.7554/eLife.18492.004 Source data for Figure 1D . DOI: http://dx.doi.org/10.7554/eLife.18492.004

    Techniques Used: Activation Assay, Staining, Western Blot, Mouse Assay, Expressing

    42) Product Images from "Expression of the Human Cytomegalovirus UL11 Glycoprotein in Viral Infection and Evaluation of Its Effect on Virus-Specific CD8 T Cells"

    Article Title: Expression of the Human Cytomegalovirus UL11 Glycoprotein in Viral Infection and Evaluation of Its Effect on Virus-Specific CD8 T Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01691-14

    Analysis of UL11 expressed in CMV-infected fibroblasts on activation of CMV-specific T cell clones. (A) CD45 expression (left) and binding of the soluble UL11-Fc protein to the indicated CD8 T cell clones (right) were determined by flow cytometry upon labeling with a CD45 Ab (black line) or an isotype control Ab (gray shading) and upon incubation with the UL11-Fc protein (black line) or the Fc control protein (gray shading). (B) Immunoblot analysis for IE1 and pp65 proteins of MRC-5 cells infected with the indicated viruses at an MOI of 2 for 78 h. GAPDH served as a loading control. (C) MRC-5 cells either mock infected or infected with the indicated viruses, as described in the legend to panel B, were examined for surface expression of HLA class I molecules by flow cytometry, and mean fluorescence intensity (MFI) values are depicted. (D) MRC-5 cells infected as described in the legend to panel B were labeled with an antibody against the major capsid protein (MCP), and nuclei were stained with DAPI. Five random images were taken per setting, and the percentages of MCP-positive cells were determined. (E) MRC-5 cells infected with the HA11G or HA11D mutant were cocultivated with CD8 T cell clones specific for the CRVLCCVML (IE1-derived) or NLVPMVATV (pp65-derived) peptide. T cell reactivity was analyzed by measuring overnight IFN-γ secretion by ELISA. Mock-infected cells and cells loaded with peptides (pep) were used as negative and positive controls, respectively. Data show the means ± SDs for triplicate samples.
    Figure Legend Snippet: Analysis of UL11 expressed in CMV-infected fibroblasts on activation of CMV-specific T cell clones. (A) CD45 expression (left) and binding of the soluble UL11-Fc protein to the indicated CD8 T cell clones (right) were determined by flow cytometry upon labeling with a CD45 Ab (black line) or an isotype control Ab (gray shading) and upon incubation with the UL11-Fc protein (black line) or the Fc control protein (gray shading). (B) Immunoblot analysis for IE1 and pp65 proteins of MRC-5 cells infected with the indicated viruses at an MOI of 2 for 78 h. GAPDH served as a loading control. (C) MRC-5 cells either mock infected or infected with the indicated viruses, as described in the legend to panel B, were examined for surface expression of HLA class I molecules by flow cytometry, and mean fluorescence intensity (MFI) values are depicted. (D) MRC-5 cells infected as described in the legend to panel B were labeled with an antibody against the major capsid protein (MCP), and nuclei were stained with DAPI. Five random images were taken per setting, and the percentages of MCP-positive cells were determined. (E) MRC-5 cells infected with the HA11G or HA11D mutant were cocultivated with CD8 T cell clones specific for the CRVLCCVML (IE1-derived) or NLVPMVATV (pp65-derived) peptide. T cell reactivity was analyzed by measuring overnight IFN-γ secretion by ELISA. Mock-infected cells and cells loaded with peptides (pep) were used as negative and positive controls, respectively. Data show the means ± SDs for triplicate samples.

    Techniques Used: Infection, Activation Assay, Expressing, Binding Assay, Flow Cytometry, Cytometry, Labeling, Incubation, Fluorescence, Staining, Mutagenesis, Clone Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay

    43) Product Images from "Rab6 Regulates Both ZW10/RINT-1- and Conserved Oligomeric Golgi Complex-dependent Golgi Trafficking and Homeostasis"

    Article Title: Rab6 Regulates Both ZW10/RINT-1- and Conserved Oligomeric Golgi Complex-dependent Golgi Trafficking and Homeostasis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E07-01-0080

    Rab6 depletion, GDP-Rab6a overexpression, or overexpression of BicD C-fragment, a Rab6 effector blocker, inhibit ZW10(102) siRNA-induced dispersal of the Golgi apparatus. (A–D) Cells double-treated with Rab6 + ZW10(102) siRNAs show compact Golgi phenotype. HeLa cells stably expressing GalNAcT2-GFP were mock (scrambled siRNA) (A) or ZW10(102) (B), Rab6 (C), and double (Rab6 + ZW10) (D) siRNA treated for 72 h and then fixed. Widefield images. (E) Total cell lysates from A to D were immunoblotted with anti-ZW10 (top row), anti-Rab 6 (middle row), and anti-tubulin (bottom row) antibodies. (F–H) Rab5 siRNA knockdown does not interfere with ZW10(102) siRNA-induced unraveling of Golgi. HeLa cells stably expressing GalNAcT2-GFP were Rab5 (F) or double (Rab5 + ZW10) (G) siRNA treated. (H) Total cell lysates from F and G were immunoblotted with anti-ZW10, anti-Rab5, and anti-tubulin as a loading control. (I) A′, GDP-Rab6a overexpression inhibited display of the siZW10 Golgi phenotype. GalNAcT2-GFP HeLa cells were transfected with ZW10 siRNA for 57 h, and then plasmids encoding GDP-Rab6 (100 ng/μl) were microinjected into cell nuclei. Six hours later, cells were fixed and stained with anti-Rab6 antibody (red). In the injected cells (red), the Golgi apparatus has a compact, juxtanuclear distribution. Strong inhibition of the ZW10 knockdown phenotype was seen in 60% of the microinjected cells. Widefield images. B′ and C′, expression of BicD C-fragment inhibits Golgi dispersal in response to ZW10 depletion. B′, control GalNAcT2-GFP HeLa cells were microinjected with plasmids (80 ng of DNA/μl stock concentration) encoding BicD C-fragment (myc tagged), and after a 24-h expression period they were myc stained (red). Expression of BicD C-fragment had no effect on the organization of the Golgi ribbon. C′, HeLa cells stably expressing GalNAcT2-GFP were incubated for 48 h after transfection with ZW10(102) siRNA. At 24 h post-siRNA transfection, cells were microinjected with plasmids (60–200 ng DNA/μl encoding BicD C-fragment; myc tagged). At the end of the 24-h expression period, cells were fixed and stained with anti-myc antibody to identify BicD-C–positive cells (red). BicD-C cells, 50% incidence, frequently showed a compact, juxtanuclear Golgi apparatus in com parison with the unraveled Golgi apparatus normally seen with ZW10 knockdown. Widefield images.
    Figure Legend Snippet: Rab6 depletion, GDP-Rab6a overexpression, or overexpression of BicD C-fragment, a Rab6 effector blocker, inhibit ZW10(102) siRNA-induced dispersal of the Golgi apparatus. (A–D) Cells double-treated with Rab6 + ZW10(102) siRNAs show compact Golgi phenotype. HeLa cells stably expressing GalNAcT2-GFP were mock (scrambled siRNA) (A) or ZW10(102) (B), Rab6 (C), and double (Rab6 + ZW10) (D) siRNA treated for 72 h and then fixed. Widefield images. (E) Total cell lysates from A to D were immunoblotted with anti-ZW10 (top row), anti-Rab 6 (middle row), and anti-tubulin (bottom row) antibodies. (F–H) Rab5 siRNA knockdown does not interfere with ZW10(102) siRNA-induced unraveling of Golgi. HeLa cells stably expressing GalNAcT2-GFP were Rab5 (F) or double (Rab5 + ZW10) (G) siRNA treated. (H) Total cell lysates from F and G were immunoblotted with anti-ZW10, anti-Rab5, and anti-tubulin as a loading control. (I) A′, GDP-Rab6a overexpression inhibited display of the siZW10 Golgi phenotype. GalNAcT2-GFP HeLa cells were transfected with ZW10 siRNA for 57 h, and then plasmids encoding GDP-Rab6 (100 ng/μl) were microinjected into cell nuclei. Six hours later, cells were fixed and stained with anti-Rab6 antibody (red). In the injected cells (red), the Golgi apparatus has a compact, juxtanuclear distribution. Strong inhibition of the ZW10 knockdown phenotype was seen in 60% of the microinjected cells. Widefield images. B′ and C′, expression of BicD C-fragment inhibits Golgi dispersal in response to ZW10 depletion. B′, control GalNAcT2-GFP HeLa cells were microinjected with plasmids (80 ng of DNA/μl stock concentration) encoding BicD C-fragment (myc tagged), and after a 24-h expression period they were myc stained (red). Expression of BicD C-fragment had no effect on the organization of the Golgi ribbon. C′, HeLa cells stably expressing GalNAcT2-GFP were incubated for 48 h after transfection with ZW10(102) siRNA. At 24 h post-siRNA transfection, cells were microinjected with plasmids (60–200 ng DNA/μl encoding BicD C-fragment; myc tagged). At the end of the 24-h expression period, cells were fixed and stained with anti-myc antibody to identify BicD-C–positive cells (red). BicD-C cells, 50% incidence, frequently showed a compact, juxtanuclear Golgi apparatus in com parison with the unraveled Golgi apparatus normally seen with ZW10 knockdown. Widefield images.

    Techniques Used: Over Expression, Stable Transfection, Expressing, Transfection, Staining, Injection, Inhibition, Concentration Assay, Incubation

    Rab6 depletion inhibits RINT-1 siRNA-induced dispersal of the Golgi apparatus. (A–D) Cell double-treated with Rab6 + RINT-1 siRNAs shows compact Golgi phenotype. HeLa cells stably expressing GalNAcT2-GFP were control (scrambled siRNA) (A) or RINT-1 (B), Rab6 (C), and double (Rab6 + RINT-1) (D) siRNA treated for 72 h and then fixed. Micrographs are maximum intensity projection of confocal image stacks. (E) Total cell lysates were immunoblotted with anti-RINT-1 (top row), anti-Rab6 (middle row), and anti-tubulin (bottom row) antibodies.
    Figure Legend Snippet: Rab6 depletion inhibits RINT-1 siRNA-induced dispersal of the Golgi apparatus. (A–D) Cell double-treated with Rab6 + RINT-1 siRNAs shows compact Golgi phenotype. HeLa cells stably expressing GalNAcT2-GFP were control (scrambled siRNA) (A) or RINT-1 (B), Rab6 (C), and double (Rab6 + RINT-1) (D) siRNA treated for 72 h and then fixed. Micrographs are maximum intensity projection of confocal image stacks. (E) Total cell lysates were immunoblotted with anti-RINT-1 (top row), anti-Rab6 (middle row), and anti-tubulin (bottom row) antibodies.

    Techniques Used: Stable Transfection, Expressing

    44) Product Images from "Expression of the Human Cytomegalovirus UL11 Glycoprotein in Viral Infection and Evaluation of Its Effect on Virus-Specific CD8 T Cells"

    Article Title: Expression of the Human Cytomegalovirus UL11 Glycoprotein in Viral Infection and Evaluation of Its Effect on Virus-Specific CD8 T Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01691-14

    Expression of ORF UL11-encoded proteins in CMV-infected cells. (A) Lysates of HM11SF- or HMpar-infected cells were subjected to precipitation with streptactin-coated agarose beads, followed by immunoblotting with a FLAG tag-specific antibody. (B, C) Lysates of HM11V5-, HM11V5S-, or HM11DL-infected RPE-1 cells, which were kept with (+) or without (−) the proteasomal inhibitor MG-132, were subjected to immunoprecipitation and immunoblotting with a V5-specific antibody. High- and low-molecular-mass UL11 bands are indicated with asterisks and closed circles, respectively. Prior to immunoprecipitation, lysates were probed with an antibody specific for the CMV UL44 protein, serving as an infection and loading control. The reactivities of the heavy chain (HC) and light chain (LC) of the V5 Ab are indicated. (D) Human fibroblasts infected with the HA11G or HA11D mutant were harvested on the indicated day postinfection (dpi) and subjected to immunoblotting with a GFP-specific (top), an IE1-specific (middle), or a UL44-specific (bottom) Ab. UL11-specific bands are indicated as described in the legend to panel A, and the nonspecific reactivity of the GFP Ab is marked with open circles. The asterisks and closed circle are as described in the legend to panels B and C. The sizes of the marker bands (indicated to the left of each panel) are in kDa.
    Figure Legend Snippet: Expression of ORF UL11-encoded proteins in CMV-infected cells. (A) Lysates of HM11SF- or HMpar-infected cells were subjected to precipitation with streptactin-coated agarose beads, followed by immunoblotting with a FLAG tag-specific antibody. (B, C) Lysates of HM11V5-, HM11V5S-, or HM11DL-infected RPE-1 cells, which were kept with (+) or without (−) the proteasomal inhibitor MG-132, were subjected to immunoprecipitation and immunoblotting with a V5-specific antibody. High- and low-molecular-mass UL11 bands are indicated with asterisks and closed circles, respectively. Prior to immunoprecipitation, lysates were probed with an antibody specific for the CMV UL44 protein, serving as an infection and loading control. The reactivities of the heavy chain (HC) and light chain (LC) of the V5 Ab are indicated. (D) Human fibroblasts infected with the HA11G or HA11D mutant were harvested on the indicated day postinfection (dpi) and subjected to immunoblotting with a GFP-specific (top), an IE1-specific (middle), or a UL44-specific (bottom) Ab. UL11-specific bands are indicated as described in the legend to panel A, and the nonspecific reactivity of the GFP Ab is marked with open circles. The asterisks and closed circle are as described in the legend to panels B and C. The sizes of the marker bands (indicated to the left of each panel) are in kDa.

    Techniques Used: Expressing, Infection, FLAG-tag, Immunoprecipitation, Mutagenesis, Marker

    45) Product Images from "Effects of tranilast on the epithelial-to-mesenchymal transition in peritoneal mesothelial cells"

    Article Title: Effects of tranilast on the epithelial-to-mesenchymal transition in peritoneal mesothelial cells

    Journal: Kidney Research and Clinical Practice

    doi: 10.23876/j.krcp.19.049

    Effects of tranilast on the Smad-dependent signaling pathway. Western blot analysis was performed on human peritoneal mesothelial cells (HPMCs) exposed to transforming growth factor-beta 1 (TGF-β1) in the presence or absence of tranilast (100 μM) for 24 hours. Quantification is relative to the control and normalized to β-actin expression. TGF-β1 increased the p-Smad2/Smad2 and p-Smad3/Smad3 protein expression ratios, increased Smad4 protein expression, and reduced Smad6 protein expression. Tranilast reversed these changes. n = 4 per group. C, control; OD, optical density; T, HPMCs with TGF-β1 treatment; T + TR, HPMCs with TGF-β1 and tranilast cotreatment. * P
    Figure Legend Snippet: Effects of tranilast on the Smad-dependent signaling pathway. Western blot analysis was performed on human peritoneal mesothelial cells (HPMCs) exposed to transforming growth factor-beta 1 (TGF-β1) in the presence or absence of tranilast (100 μM) for 24 hours. Quantification is relative to the control and normalized to β-actin expression. TGF-β1 increased the p-Smad2/Smad2 and p-Smad3/Smad3 protein expression ratios, increased Smad4 protein expression, and reduced Smad6 protein expression. Tranilast reversed these changes. n = 4 per group. C, control; OD, optical density; T, HPMCs with TGF-β1 treatment; T + TR, HPMCs with TGF-β1 and tranilast cotreatment. * P

    Techniques Used: Western Blot, Expressing

    Effects of tranilast on the Akt/β-catenin signaling pathway. Western blot analysis was performed on human peritoneal mesothelial cells (HPMCs) exposed to transforming growth factor-beta 1 (TGF-β1) in the presence or absence of tranilast (100 μM) for 24 hours. Quantification is relative to the control and normalized to β-actin expression. TGF-β1 increased phosphorylated-Akt (p-Akt)/Akt protein expression ratio and β-catenin protein expression. Tranilast reversed these changes. n = 4 per group. C, control; OD, optical density; T, HPMCs with TGF-β1 treatment; T + TR, HPMCs with TGF-β1 and tranilast cotreatment. * P
    Figure Legend Snippet: Effects of tranilast on the Akt/β-catenin signaling pathway. Western blot analysis was performed on human peritoneal mesothelial cells (HPMCs) exposed to transforming growth factor-beta 1 (TGF-β1) in the presence or absence of tranilast (100 μM) for 24 hours. Quantification is relative to the control and normalized to β-actin expression. TGF-β1 increased phosphorylated-Akt (p-Akt)/Akt protein expression ratio and β-catenin protein expression. Tranilast reversed these changes. n = 4 per group. C, control; OD, optical density; T, HPMCs with TGF-β1 treatment; T + TR, HPMCs with TGF-β1 and tranilast cotreatment. * P

    Techniques Used: Western Blot, Expressing

    Western blot analysis of epithelial-to-mesenchymal transition markers in human peritoneal mesothelial cells (HPMCs). HPMCs were treated with transforming growth factor-beta 1 (TGF-β1) in the presence or absence of tranilast (100 μM) for 24 hours. Quantification is relative to the control and normalized to β-actin expression. TGF-β1 increased the protein expression of α-smooth muscle actin (α-SMA) (A) and Snail (B) and reduced the protein expression of E-cadherin (A) in HPMCs. Tranilast reversed the changes in these markers. n = 4 per group. C, control; OD, optical density; T, HPMCs with TGF-β1 treatment; T + TR, HPMCs with TGF-β1 and tranilast cotreatment. * P
    Figure Legend Snippet: Western blot analysis of epithelial-to-mesenchymal transition markers in human peritoneal mesothelial cells (HPMCs). HPMCs were treated with transforming growth factor-beta 1 (TGF-β1) in the presence or absence of tranilast (100 μM) for 24 hours. Quantification is relative to the control and normalized to β-actin expression. TGF-β1 increased the protein expression of α-smooth muscle actin (α-SMA) (A) and Snail (B) and reduced the protein expression of E-cadherin (A) in HPMCs. Tranilast reversed the changes in these markers. n = 4 per group. C, control; OD, optical density; T, HPMCs with TGF-β1 treatment; T + TR, HPMCs with TGF-β1 and tranilast cotreatment. * P

    Techniques Used: Western Blot, Expressing

    Effects of tranilast on transforming growth factor-beta 1 (TGF-β1)–induced morphological changes. The effects of tranilast on TGF-β1-induced changes in human peritoneal mesothelial cell (HPMC) morphology were observed by phase-contrast microscopy. HPMCs treated with control buffer or tranilast exhibited a cobblestone appearance (C group), while those exposed to TGF-β1 exhibited a spindle shape (T group). HPMCs exposed to TGF-β1 and tranilast reverted to a cobblestone appearance (T + TR1, T + TR10, and T + TR100 groups). C, control; OD, optical density; T, HPMCs exposed to TGF-β1; T + TR1, HPMCs exposed to TGF-β1 and tranilast 1 μM; T + TR10, HPMCs exposed to TGF-β1 and tranilast 10 μM; T + TR100, HPMCs exposed to TGF-β1 and tranilast 100 μM; TR100, HPMCs exposed to tranilast 100 μM.
    Figure Legend Snippet: Effects of tranilast on transforming growth factor-beta 1 (TGF-β1)–induced morphological changes. The effects of tranilast on TGF-β1-induced changes in human peritoneal mesothelial cell (HPMC) morphology were observed by phase-contrast microscopy. HPMCs treated with control buffer or tranilast exhibited a cobblestone appearance (C group), while those exposed to TGF-β1 exhibited a spindle shape (T group). HPMCs exposed to TGF-β1 and tranilast reverted to a cobblestone appearance (T + TR1, T + TR10, and T + TR100 groups). C, control; OD, optical density; T, HPMCs exposed to TGF-β1; T + TR1, HPMCs exposed to TGF-β1 and tranilast 1 μM; T + TR10, HPMCs exposed to TGF-β1 and tranilast 10 μM; T + TR100, HPMCs exposed to TGF-β1 and tranilast 100 μM; TR100, HPMCs exposed to tranilast 100 μM.

    Techniques Used: Microscopy

    46) Product Images from "Neural Mechanisms Underlying the Disruption of Male Courtship Behavior by Adult Exposure to Di(2-ethylhexyl) Phthalate in Mice"

    Article Title: Neural Mechanisms Underlying the Disruption of Male Courtship Behavior by Adult Exposure to Di(2-ethylhexyl) Phthalate in Mice

    Journal: Environmental Health Perspectives

    doi: 10.1289/EHP1443

    Validation of proteomic data and characterization of androgen receptor (AR) and estrogen receptor ( ER ) α expression in the medial preoptic nucleus (MPN). ( A ) 3-D view of the spot ID:0656, corresponding to GFAP, shown for three animals exposed to the vehicle (Veh) or DEHP at 5 or 50 μ g / kg / d . ( B – C ) Upper panels: Representative Western blots of GFAP ( B ), β - actin ( C ), and GAPDH used as a protein reference, in the MPN of the Veh, DEHP-5, and DEHP-50 groups. Lower panels: quantification of the protein levels normalized to GAPDH. Data are expressed as the means ± SEM of four males per treatment group, * p
    Figure Legend Snippet: Validation of proteomic data and characterization of androgen receptor (AR) and estrogen receptor ( ER ) α expression in the medial preoptic nucleus (MPN). ( A ) 3-D view of the spot ID:0656, corresponding to GFAP, shown for three animals exposed to the vehicle (Veh) or DEHP at 5 or 50 μ g / kg / d . ( B – C ) Upper panels: Representative Western blots of GFAP ( B ), β - actin ( C ), and GAPDH used as a protein reference, in the MPN of the Veh, DEHP-5, and DEHP-50 groups. Lower panels: quantification of the protein levels normalized to GAPDH. Data are expressed as the means ± SEM of four males per treatment group, * p

    Techniques Used: Expressing, Western Blot

    47) Product Images from "Premature Aging in Skeletal Muscle Lacking Serum Response Factor"

    Article Title: Premature Aging in Skeletal Muscle Lacking Serum Response Factor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003910

    Age-dependant down-regulation of SRF expression in skeletal muscle of mice and human. (A) Immunoblot analysis of protein extracts from 2, 10 and 15 month-old control muscles using anti-SRF antibody. Anti-α-Tubulin is used as a loading control. (B) Quantitative real-time PCR was performed on RNAs prepared from Gastrocnemius muscles of control mice 2 (white) and 15 (grey) months old. Mean expression levels for SRF , Skeletal and Cardiac α-actin mRNAs was normalized using Cyclophilin transcript as reference. (n = 4). *P
    Figure Legend Snippet: Age-dependant down-regulation of SRF expression in skeletal muscle of mice and human. (A) Immunoblot analysis of protein extracts from 2, 10 and 15 month-old control muscles using anti-SRF antibody. Anti-α-Tubulin is used as a loading control. (B) Quantitative real-time PCR was performed on RNAs prepared from Gastrocnemius muscles of control mice 2 (white) and 15 (grey) months old. Mean expression levels for SRF , Skeletal and Cardiac α-actin mRNAs was normalized using Cyclophilin transcript as reference. (n = 4). *P

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    48) Product Images from "Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿ †"

    Article Title: Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.02063-08

    T57 phosphorylation increases the stability and abundance of full-length 16E1^E4 in the cell. (A) His-16E1^E4 was incubated at 30°C in the presence (+) or absence (−) of ERK for 1 h and analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4 (phospho T57). High- and low-level exposures of these blots are shown, demonstrating that the phosphospecific antibody did not detect unphosphorylated E1^E4 (upper panels). SiHa or PHK cells were infected with rAd16E1^E4 for 24 h, and cell lysates were analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4. The antibody to T57-phosphorylated 16E1^E4 detects only the slower-migrating band, confirming that this band is the T57-phosphorylated form of 16E1^E4 (lower panels). (B) SiHa cells were infected with rAd16E1^E4 for 24 or 48 h, fixed with 5% formaldehyde, and triple stained with antibodies to total 16E1^E4 (green) and T57-phosphorylated 16E1^E4 (red) and with DAPI (4′,6-diamidino-2-phenylindole; blue). T57-phosphorylated 16E1^E4 appeared only in cells containing abundant E1^E4 (shown by arrows). Images were captured using a 20× objective. (C) SiHa cells were infected with rAd16E1^E4 for 24 h, and then the SiHa cells expressing high and low levels of E1^E4 were separated by FACS and analyzed by Western blotting with antibodies against 16E1^E4 and GAPDH (as a loading control). Fluorescence intensity correlated with protein abundance and the presence of the T57-phosphorylated (slower-migrating) E1^E4 form. The molecular masses of 16E^E4 forms (in kilodaltons) are shown to the right of the lower panel. +ve, positive; −ve, negative; FITC, fluorescein isothiocyanate. (D, top panel) SiHa cells were transfected with a 16E1^E4 WT-, T57A-, or T57D-expressing plasmid for 24 h prior to SDS-PAGE and Western blotting to reveal the different migration patterns. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the left. (Bottom panels) SiHa cells were transfected with a 16E1^E4 WT- or T57D-expressing plasmid for 24 h before being treated with 40 μg/ml cycloheximide for 0, 2, 4, 6, 12, or 24 h prior to harvest. Equal volumes of the soluble protein fraction were analyzed by Western blotting to show the increased stability of 16E1^E4 T57D relative to those of the non-T57-phosphorylated WT 16E1^E4 and the GAPDH loading controls. The results shown are typical of results from triplicate experiments. (E) SiHa cells were cotransfected with a plasmid expressing either T57A, T57D, or WT 16E1^E4 and the GFP-expressing plasmid pXJ-GFP (as a transfection control). Cells were harvested at multiple time points posttransfection, as indicated. Total extracts were run on SDS-polyacrylamide gels and analyzed with antibodies against GFP and 16E1^E4 to show the accumulation of 16E1^E4 in the cell. The time course shows the more rapid accumulation of the T57D (phosphomimic) form than of the WT 16E1^E4 (which lacks a T57-phosphorylated form before the 24-h time point) and the 16E1^E4 T57A mutant (which lacks the T57 phosphorylation site). The results shown are typical of results from triplicate experiments. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the right (left panel).
    Figure Legend Snippet: T57 phosphorylation increases the stability and abundance of full-length 16E1^E4 in the cell. (A) His-16E1^E4 was incubated at 30°C in the presence (+) or absence (−) of ERK for 1 h and analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4 (phospho T57). High- and low-level exposures of these blots are shown, demonstrating that the phosphospecific antibody did not detect unphosphorylated E1^E4 (upper panels). SiHa or PHK cells were infected with rAd16E1^E4 for 24 h, and cell lysates were analyzed by Western blotting using antibodies to 16E1^E4 or to T57-phosphorylated 16E1^E4. The antibody to T57-phosphorylated 16E1^E4 detects only the slower-migrating band, confirming that this band is the T57-phosphorylated form of 16E1^E4 (lower panels). (B) SiHa cells were infected with rAd16E1^E4 for 24 or 48 h, fixed with 5% formaldehyde, and triple stained with antibodies to total 16E1^E4 (green) and T57-phosphorylated 16E1^E4 (red) and with DAPI (4′,6-diamidino-2-phenylindole; blue). T57-phosphorylated 16E1^E4 appeared only in cells containing abundant E1^E4 (shown by arrows). Images were captured using a 20× objective. (C) SiHa cells were infected with rAd16E1^E4 for 24 h, and then the SiHa cells expressing high and low levels of E1^E4 were separated by FACS and analyzed by Western blotting with antibodies against 16E1^E4 and GAPDH (as a loading control). Fluorescence intensity correlated with protein abundance and the presence of the T57-phosphorylated (slower-migrating) E1^E4 form. The molecular masses of 16E^E4 forms (in kilodaltons) are shown to the right of the lower panel. +ve, positive; −ve, negative; FITC, fluorescein isothiocyanate. (D, top panel) SiHa cells were transfected with a 16E1^E4 WT-, T57A-, or T57D-expressing plasmid for 24 h prior to SDS-PAGE and Western blotting to reveal the different migration patterns. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the left. (Bottom panels) SiHa cells were transfected with a 16E1^E4 WT- or T57D-expressing plasmid for 24 h before being treated with 40 μg/ml cycloheximide for 0, 2, 4, 6, 12, or 24 h prior to harvest. Equal volumes of the soluble protein fraction were analyzed by Western blotting to show the increased stability of 16E1^E4 T57D relative to those of the non-T57-phosphorylated WT 16E1^E4 and the GAPDH loading controls. The results shown are typical of results from triplicate experiments. (E) SiHa cells were cotransfected with a plasmid expressing either T57A, T57D, or WT 16E1^E4 and the GFP-expressing plasmid pXJ-GFP (as a transfection control). Cells were harvested at multiple time points posttransfection, as indicated. Total extracts were run on SDS-polyacrylamide gels and analyzed with antibodies against GFP and 16E1^E4 to show the accumulation of 16E1^E4 in the cell. The time course shows the more rapid accumulation of the T57D (phosphomimic) form than of the WT 16E1^E4 (which lacks a T57-phosphorylated form before the 24-h time point) and the 16E1^E4 T57A mutant (which lacks the T57 phosphorylation site). The results shown are typical of results from triplicate experiments. The molecular masses of 16E1^E4 forms (in kilodaltons) are shown to the right (left panel).

    Techniques Used: Incubation, Western Blot, Infection, Staining, Expressing, FACS, Fluorescence, Transfection, Plasmid Preparation, SDS Page, Migration, Mutagenesis

    16E1^E4 protein is phosphorylated by multiple kinases, with ERK stimulating a gel shift. (A) Putative phosphorylation sites of 16E1^E4 based on common kinase consensus sites are shown. (B) His-16E1^E4 was used as a substrate in kinase assays with [γ- 32 P]ATP and CDK1, CDK2, CKII, ERK, PKA, or PKC α. Samples were separated by SDS-PAGE, and then phosphorylation was detected using a phosphorimager. His-16E1^E4 was phosphorylated by CDK1, CDK2, ERK, PKA, and PKC α. Autophosphorylation (auto-phospho) of the kinase is apparent in the CKII and PKC α lanes. Molecular mass standards (in kilodaltons) are shown to the right. 16E1^E4 phospho, phosphorylated 16E1^E4; +, present; −, absent. (C) His-16E1^E4 was incubated with or without ERK under kinase assay conditions and then analyzed by silver staining. 16E1^E4 phosphorylation by ERK caused a gel shift. Molecular mass standards (in kilodaltons) are shown to the left. (D) rAd16E1^E4-infected SiHa cells were incubated with the kinase inhibitors SB203580, PD98059, and U0126 (separately or in combination) 6 h postinfection. Cells were harvested 24 h postinfection, and extracts were analyzed by Western blotting with anti-16E1^E4 antibody. The MEK inhibitors PD98059 and/or U0126 reduced the intensity of the upper 16E1^E4 band.
    Figure Legend Snippet: 16E1^E4 protein is phosphorylated by multiple kinases, with ERK stimulating a gel shift. (A) Putative phosphorylation sites of 16E1^E4 based on common kinase consensus sites are shown. (B) His-16E1^E4 was used as a substrate in kinase assays with [γ- 32 P]ATP and CDK1, CDK2, CKII, ERK, PKA, or PKC α. Samples were separated by SDS-PAGE, and then phosphorylation was detected using a phosphorimager. His-16E1^E4 was phosphorylated by CDK1, CDK2, ERK, PKA, and PKC α. Autophosphorylation (auto-phospho) of the kinase is apparent in the CKII and PKC α lanes. Molecular mass standards (in kilodaltons) are shown to the right. 16E1^E4 phospho, phosphorylated 16E1^E4; +, present; −, absent. (C) His-16E1^E4 was incubated with or without ERK under kinase assay conditions and then analyzed by silver staining. 16E1^E4 phosphorylation by ERK caused a gel shift. Molecular mass standards (in kilodaltons) are shown to the left. (D) rAd16E1^E4-infected SiHa cells were incubated with the kinase inhibitors SB203580, PD98059, and U0126 (separately or in combination) 6 h postinfection. Cells were harvested 24 h postinfection, and extracts were analyzed by Western blotting with anti-16E1^E4 antibody. The MEK inhibitors PD98059 and/or U0126 reduced the intensity of the upper 16E1^E4 band.

    Techniques Used: Electrophoretic Mobility Shift Assay, SDS Page, Incubation, Kinase Assay, Silver Staining, Infection, Western Blot

    The ERK-mediated gel shift is triggered by the phosphorylation of the threonine residue at consensus site position 57. (A) The His-16E^E4 WT and mutants S43/44A, S49A, T51A, T54A, and T57A were used in nonradioactive kinase assays and detected by SDS-PAGE and silver staining. Only 16E1^E4 T57A (with a mutation in the ERK/MAPK consensus site) failed to show the gel shift. A molecular mass standard (in kilodaltons) is shown to the left. +, present; −, absent. (B) 2D SDS-PAGE and Western blotting show that in the cells transfected with WT or mutant 16E1^E4, WT 16E1^E4 exists as an unphosphorylated protein (pI 9.2) and a singly phosphorylated form (pI 8). A minor multiply phosphorylated species (pI 6.7) increases in abundance in the presence of OA. In the presence of OA (PP2A inhibition), the T57A and S32A mutants, but not mutant S43/44A, failed to produce the second main spot. (C) His-16E1^E4 WT and S32A proteins were analyzed by nonradioactive kinase assays and detected by SDS-PAGE and silver staining. S32A phosphorylation by CDK1 was abolished, and that by CDK2 was clearly reduced. A molecular mass standard (in kilodaltons) is shown to the left.
    Figure Legend Snippet: The ERK-mediated gel shift is triggered by the phosphorylation of the threonine residue at consensus site position 57. (A) The His-16E^E4 WT and mutants S43/44A, S49A, T51A, T54A, and T57A were used in nonradioactive kinase assays and detected by SDS-PAGE and silver staining. Only 16E1^E4 T57A (with a mutation in the ERK/MAPK consensus site) failed to show the gel shift. A molecular mass standard (in kilodaltons) is shown to the left. +, present; −, absent. (B) 2D SDS-PAGE and Western blotting show that in the cells transfected with WT or mutant 16E1^E4, WT 16E1^E4 exists as an unphosphorylated protein (pI 9.2) and a singly phosphorylated form (pI 8). A minor multiply phosphorylated species (pI 6.7) increases in abundance in the presence of OA. In the presence of OA (PP2A inhibition), the T57A and S32A mutants, but not mutant S43/44A, failed to produce the second main spot. (C) His-16E1^E4 WT and S32A proteins were analyzed by nonradioactive kinase assays and detected by SDS-PAGE and silver staining. S32A phosphorylation by CDK1 was abolished, and that by CDK2 was clearly reduced. A molecular mass standard (in kilodaltons) is shown to the left.

    Techniques Used: Electrophoretic Mobility Shift Assay, SDS Page, Silver Staining, Mutagenesis, Western Blot, Transfection, Inhibition

    T57 phosphorylation triggers 16E1^E4 structural change. (A) Dependence of electrophoretic mobilities ( R m ) of the 16E1^E4 phosphorylated (E4-p) and unphosphorylated (E4) species on the acrylamide concentration in SDS-PAGE (Ferguson plot). (B) Intrinsic fluorescence upon the phosphorylation of His-16E1^E4 by ERK was monitored. The fluorescence maxima prior to phosphorylation (time, 0 min; green) and upon the completion of phosphorylation (time, 30 min; red) are indicated on the plot. The decrease in fluorescence intensity and the red shift of the spectrum accompanying phosphorylation are consistent with the tryptophan residues becoming more buried in a hydrophobic environment. (C) The 16E1^E4 protein has elements of secondary structure at its C and N termini (red, α helix; blue, β sheet). T57 is located in a highly charged unstructured region of the protein, and its phosphorylation contributes to the polarization of this region. The effect of T57 phosphorylation on this unstructured region of the protein was assessed using a 27-amino-acid peptide as indicated. (D) The effect of T57 phosphorylation on the structure of the peptide (indicated in panel C) was monitored by recording fluorescent resonance energy transfer (FRET) between the tryptophan residue and a fluorescence acceptor molecule on the terminal cysteine. The excitation (250- to 400-nm) and emission (450- to 600-nm) spectra of the phosphorylated (E4-P) and unphosphorylated (E4) peptides are shown. The peak at 280 nm in the difference spectrum reveals that fluorescence transfer is observed only in the phosphorylated peptide, which indicates that T57 phosphorylation compacts the peptide. (E) Schematic summarizing the effect of T57 phosphorylation by ERK on the structure of 16E1^E4. Upon phosphorylation, the addition of a negative charge on T57 increases the charge polarization in the loop region of the protein and, as indicated by the findings of the tryptophan fluorescence studies, results in additional restraints in this region.
    Figure Legend Snippet: T57 phosphorylation triggers 16E1^E4 structural change. (A) Dependence of electrophoretic mobilities ( R m ) of the 16E1^E4 phosphorylated (E4-p) and unphosphorylated (E4) species on the acrylamide concentration in SDS-PAGE (Ferguson plot). (B) Intrinsic fluorescence upon the phosphorylation of His-16E1^E4 by ERK was monitored. The fluorescence maxima prior to phosphorylation (time, 0 min; green) and upon the completion of phosphorylation (time, 30 min; red) are indicated on the plot. The decrease in fluorescence intensity and the red shift of the spectrum accompanying phosphorylation are consistent with the tryptophan residues becoming more buried in a hydrophobic environment. (C) The 16E1^E4 protein has elements of secondary structure at its C and N termini (red, α helix; blue, β sheet). T57 is located in a highly charged unstructured region of the protein, and its phosphorylation contributes to the polarization of this region. The effect of T57 phosphorylation on this unstructured region of the protein was assessed using a 27-amino-acid peptide as indicated. (D) The effect of T57 phosphorylation on the structure of the peptide (indicated in panel C) was monitored by recording fluorescent resonance energy transfer (FRET) between the tryptophan residue and a fluorescence acceptor molecule on the terminal cysteine. The excitation (250- to 400-nm) and emission (450- to 600-nm) spectra of the phosphorylated (E4-P) and unphosphorylated (E4) peptides are shown. The peak at 280 nm in the difference spectrum reveals that fluorescence transfer is observed only in the phosphorylated peptide, which indicates that T57 phosphorylation compacts the peptide. (E) Schematic summarizing the effect of T57 phosphorylation by ERK on the structure of 16E1^E4. Upon phosphorylation, the addition of a negative charge on T57 increases the charge polarization in the loop region of the protein and, as indicated by the findings of the tryptophan fluorescence studies, results in additional restraints in this region.

    Techniques Used: Concentration Assay, SDS Page, Fluorescence, Förster Resonance Energy Transfer

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    Centrifugation:

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: Cells were harvested with RIPA lysis buffer (Pierce, USA) containing anti-protease cocktail (Roche, USA) and cleared by centrifugation at 4°C. .. The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    MTT Assay:

    Article Title: Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species
    Article Snippet: .. MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide), GSH, 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), thiobarbituric acid (TBA), 2,7-dichlorofluorescin diacetate (DCFH-DA), anti-p53 antibody, anti-bax antibody, anti-bcl-2 antibody, and anti-β-actin antibody were obtained from Sigma-Aldrich (St Louis, MO). .. Secondary antibodies, RIPA buffer, and sodium dodecyl sulfate (SDS) were bought from Santa Cruz Biotechnology, Inc, (Santa Cruz, CA).

    Cytometry:

    Article Title: Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis
    Article Snippet: .. For flow cytometry experiments, treated cells were fixed in 4% formalin for 15 min and stained with conformation-specific primary antibodies (Bax 6A7 [Sigma, no. B8429] and Bak NT [Millipore, no. 06536]). .. Species-specific Alexa fluor 647 or Cy5-conjugated secondary antibodies were used to detect activated Bax and Bak by flow cytometry.

    Blocking Assay:

    Article Title: Safflor yellow B reduces hypoxia-mediated vasoconstriction by regulating endothelial micro ribonucleic acid/nitric oxide synthase signaling
    Article Snippet: .. After blocking with 1% bovine serum albumin for 120 min at 20 ± 2°C, the membranes were incubated with one of the following primary antibodies: anti-HIF-1α (1:1000, ab216842), anti-caspase 3 (1:500, ab32042), anti-eNOS (1:500, ab95254), anti-p-eNOSSer1177 (1:500, ab51038, all Abcam), anti-Bcl-2 (1:1000, SAB4500003), anti-Bax (1:1000, SAB4502546), anti-iNOS (1:1000, SAB4502011), anti-p-iNOSTyr151 (1:1000, SAB4301563), and anti-S100A1 (1:250, SAB4502708, all Sigma-Aldrich) overnight at 4°C prior to washing thrice (15-min each) with Tris-buffered saline containing Tween 20 (TBST). .. The blots were then incubated with the corresponding HRP-conjugated secondary antibody for 50–60 min at 20 ± 2°C, followed by three 10-min washes with TBST.

    Electrophoresis:

    Article Title: Safflor yellow B reduces hypoxia-mediated vasoconstriction by regulating endothelial micro ribonucleic acid/nitric oxide synthase signaling
    Article Snippet: Equal amounts of total proteins (40 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel (15%) electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane (IPVH00010, Millipore, Bedford, MA, USA). .. After blocking with 1% bovine serum albumin for 120 min at 20 ± 2°C, the membranes were incubated with one of the following primary antibodies: anti-HIF-1α (1:1000, ab216842), anti-caspase 3 (1:500, ab32042), anti-eNOS (1:500, ab95254), anti-p-eNOSSer1177 (1:500, ab51038, all Abcam), anti-Bcl-2 (1:1000, SAB4500003), anti-Bax (1:1000, SAB4502546), anti-iNOS (1:1000, SAB4502011), anti-p-iNOSTyr151 (1:1000, SAB4301563), and anti-S100A1 (1:250, SAB4502708, all Sigma-Aldrich) overnight at 4°C prior to washing thrice (15-min each) with Tris-buffered saline containing Tween 20 (TBST).

    Incubation:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: .. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK). .. The membranes were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody.

    Article Title: Psoralea glandulosa as a Potential Source of Anticancer Agents for Melanoma Treatment
    Article Snippet: .. Bcl-2 (SAB2500154, Sigma Aldrich, Milan, Italy) (1:500 dilution), Bax (B3428, Sigma-Aldrich) (1:2000 dilution), rabbit polyclonal anti-p53 (FL-393; sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:300 dilution) and α-tubulin (T5326; Sigma-Aldrich) (1:5000 dilution) antibodies were diluted in TBST and membranes incubated for 2 h at room temperature. .. Antibodies were detected with horseradish peroxidase-conjugated secondary antibody using the enhanced chemiluminescence detection Supersignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL, USA).

    Article Title: Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer
    Article Snippet: Cell lysates were incubated with primary antibodies for 3 h at 4 °C. .. For immunoprecipitation, MCL-1 antibody (BD Biosciences, Cat # 554103) and conformation-specific antibodies for BAK (Millipore, Cat # 06-536) or BAX (Sigma, Cat # B8429) was used as primary antibody.

    Article Title: Involvement of Bax and Bcl-2 in Induction of Apoptosis by Essential Oils of Three Lebanese Salvia Species in Human Prostate Cancer Cells
    Article Snippet: .. Bcl-2 (SAB2500154, Sigma Aldrich) (1:500 dilution), -Bax (B3428, Sigma Aldrich) (1:2000 dilution), -cleaved caspase-9 (AB3629, Sigma Aldrich) (1:500 dilution), and α-tubulin (T5326; Sigma Aldrich) (1:5000 dilution) antibodies were diluted in Tris Buffered Saline, 0.1% Tween 20 (TBST)and membranes incubated for 2 h at room temperature. .. Antibodies were detected with horseradish peroxidase-conjugated secondary antibody using the enhanced chemiluminescence detection Supersignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL, USA).

    Article Title: Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis
    Article Snippet: After incubation, the reaction was stopped by adding 1 mM DTT for an additional 15 min at room temperature. .. For flow cytometry experiments, treated cells were fixed in 4% formalin for 15 min and stained with conformation-specific primary antibodies (Bax 6A7 [Sigma, no. B8429] and Bak NT [Millipore, no. 06536]).

    Article Title: Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death
    Article Snippet: .. 500–1000 μ g protein was immunoprecipitated and incubated overnight at 4 °C with 2 μ g/ml mouse anti-BAK antibody (Ab-1; Merck Millipore, Billerica, MA, USA) or anti-BAX antibody (6A7, Sigma-Aldrich) and 10 μ l pan-mouse IgG Dynabeads (Life technologies, Inc.), washed with CHAPS lysis buffer and analyzed by western blotting using rabbit anti-BAK antibody (BD Bioscience) or anti-BAX antibody (Merck, Darmstadt, Germany). .. Immunoprecipitation of MCL-1 was performed in 500 μ l lysates containing up to 1000 μ g proteins, which were incubated overnight at 4 °C with 2 μ g/ml mouse anti-MCL-1 antibody (BD Biosciences) and 10 μ l pan-mouse IgG Dynabeads or Protein G Dynabeads (Life Technologies, Inc.) and washed with CHAPS buffer.

    Article Title: Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p, et al. Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p
    Article Snippet: Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl‐2 (SAB4500005, 1:1000) from Sigma‐Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C. .. The next day, the membrane was incubated with horseradish peroxidase (HRP)–labelled goat anti‐rabbit IgG (ab6721, 1:20 000) or goat antimouse IgG (ab6789, 1:10 000) from Abcam for 1 h at room temperature.

    Article Title: Safflor yellow B reduces hypoxia-mediated vasoconstriction by regulating endothelial micro ribonucleic acid/nitric oxide synthase signaling
    Article Snippet: .. After blocking with 1% bovine serum albumin for 120 min at 20 ± 2°C, the membranes were incubated with one of the following primary antibodies: anti-HIF-1α (1:1000, ab216842), anti-caspase 3 (1:500, ab32042), anti-eNOS (1:500, ab95254), anti-p-eNOSSer1177 (1:500, ab51038, all Abcam), anti-Bcl-2 (1:1000, SAB4500003), anti-Bax (1:1000, SAB4502546), anti-iNOS (1:1000, SAB4502011), anti-p-iNOSTyr151 (1:1000, SAB4301563), and anti-S100A1 (1:250, SAB4502708, all Sigma-Aldrich) overnight at 4°C prior to washing thrice (15-min each) with Tris-buffered saline containing Tween 20 (TBST). .. The blots were then incubated with the corresponding HRP-conjugated secondary antibody for 50–60 min at 20 ± 2°C, followed by three 10-min washes with TBST.

    Expressing:

    Article Title: Psoralea glandulosa as a Potential Source of Anticancer Agents for Melanoma Treatment
    Article Snippet: Western Blot Analysis The expression of Bcl-2, Bax, and p53 proteins was evaluated by Western blot analysis. .. Bcl-2 (SAB2500154, Sigma Aldrich, Milan, Italy) (1:500 dilution), Bax (B3428, Sigma-Aldrich) (1:2000 dilution), rabbit polyclonal anti-p53 (FL-393; sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:300 dilution) and α-tubulin (T5326; Sigma-Aldrich) (1:5000 dilution) antibodies were diluted in TBST and membranes incubated for 2 h at room temperature.

    Article Title: Involvement of Bax and Bcl-2 in Induction of Apoptosis by Essential Oils of Three Lebanese Salvia Species in Human Prostate Cancer Cells
    Article Snippet: Western Blot Analysis The expression of Bcl-2, Bax and cleaved caspase-9 proteins was evaluated by western blot analysis, as previously described [ ]. .. Bcl-2 (SAB2500154, Sigma Aldrich) (1:500 dilution), -Bax (B3428, Sigma Aldrich) (1:2000 dilution), -cleaved caspase-9 (AB3629, Sigma Aldrich) (1:500 dilution), and α-tubulin (T5326; Sigma Aldrich) (1:5000 dilution) antibodies were diluted in Tris Buffered Saline, 0.1% Tween 20 (TBST)and membranes incubated for 2 h at room temperature.

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich). .. The expression levels of investigated proteins were determined and normalized to the internal control, β-actin.

    Article Title: Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p, et al. Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p
    Article Snippet: Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl‐2 (SAB4500005, 1:1000) from Sigma‐Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C. .. The protein expression was analysed using the ImageJ software (National Institutes of Health).GAPDH was used as an internal control.

    BIA-KA:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: The protein concentration was measured by a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions. .. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK).

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: Protein concentrations were measured by BCA reagents (Pierce). .. The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Western Blot:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: Paragraph title: Western blot analysis ... Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK).

    Article Title: Psoralea glandulosa as a Potential Source of Anticancer Agents for Melanoma Treatment
    Article Snippet: Paragraph title: 3.11. Western Blot Analysis ... Bcl-2 (SAB2500154, Sigma Aldrich, Milan, Italy) (1:500 dilution), Bax (B3428, Sigma-Aldrich) (1:2000 dilution), rabbit polyclonal anti-p53 (FL-393; sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:300 dilution) and α-tubulin (T5326; Sigma-Aldrich) (1:5000 dilution) antibodies were diluted in TBST and membranes incubated for 2 h at room temperature.

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: Paragraph title: Western blot ... Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour.

    Article Title: Involvement of Bax and Bcl-2 in Induction of Apoptosis by Essential Oils of Three Lebanese Salvia Species in Human Prostate Cancer Cells
    Article Snippet: Paragraph title: 4.2.7. Western Blot Analysis ... Bcl-2 (SAB2500154, Sigma Aldrich) (1:500 dilution), -Bax (B3428, Sigma Aldrich) (1:2000 dilution), -cleaved caspase-9 (AB3629, Sigma Aldrich) (1:500 dilution), and α-tubulin (T5326; Sigma Aldrich) (1:5000 dilution) antibodies were diluted in Tris Buffered Saline, 0.1% Tween 20 (TBST)and membranes incubated for 2 h at room temperature.

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: Paragraph title: Western blot analysis ... The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Article Title: Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death
    Article Snippet: .. 500–1000 μ g protein was immunoprecipitated and incubated overnight at 4 °C with 2 μ g/ml mouse anti-BAK antibody (Ab-1; Merck Millipore, Billerica, MA, USA) or anti-BAX antibody (6A7, Sigma-Aldrich) and 10 μ l pan-mouse IgG Dynabeads (Life technologies, Inc.), washed with CHAPS lysis buffer and analyzed by western blotting using rabbit anti-BAK antibody (BD Bioscience) or anti-BAX antibody (Merck, Darmstadt, Germany). .. Immunoprecipitation of MCL-1 was performed in 500 μ l lysates containing up to 1000 μ g proteins, which were incubated overnight at 4 °C with 2 μ g/ml mouse anti-MCL-1 antibody (BD Biosciences) and 10 μ l pan-mouse IgG Dynabeads or Protein G Dynabeads (Life Technologies, Inc.) and washed with CHAPS buffer.

    Article Title: Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p, et al. Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p
    Article Snippet: Paragraph title: Western blot analysis ... Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl‐2 (SAB4500005, 1:1000) from Sigma‐Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C.

    Article Title: Recombinant VP1, an Akt Inhibitor, Suppresses Progression of Hepatocellular Carcinoma by Inducing Apoptosis and Modulation of CCL2 Production
    Article Snippet: Immunoprecipitation For detection of Ku70-Bax interactions in BNL and Hepa1-6 cells, cells treated with or without rVP1 were harvested in protein extraction reagents (Pierce), and 0.2–1 mg of cell lysates were immunoprecipitated with 4–10 µg of anti-Bax antibody (Sigma). .. After washing with PBS five times, western blot analysis of pre-immunoprecipitated (Input) and immunoprecipitated (IP) samples was performed with an anti-Ku70 antibody.

    Article Title: Safflor yellow B reduces hypoxia-mediated vasoconstriction by regulating endothelial micro ribonucleic acid/nitric oxide synthase signaling
    Article Snippet: Paragraph title: Western blot analysis ... After blocking with 1% bovine serum albumin for 120 min at 20 ± 2°C, the membranes were incubated with one of the following primary antibodies: anti-HIF-1α (1:1000, ab216842), anti-caspase 3 (1:500, ab32042), anti-eNOS (1:500, ab95254), anti-p-eNOSSer1177 (1:500, ab51038, all Abcam), anti-Bcl-2 (1:1000, SAB4500003), anti-Bax (1:1000, SAB4502546), anti-iNOS (1:1000, SAB4502011), anti-p-iNOSTyr151 (1:1000, SAB4301563), and anti-S100A1 (1:250, SAB4502708, all Sigma-Aldrich) overnight at 4°C prior to washing thrice (15-min each) with Tris-buffered saline containing Tween 20 (TBST).

    Flow Cytometry:

    Article Title: Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis
    Article Snippet: .. For flow cytometry experiments, treated cells were fixed in 4% formalin for 15 min and stained with conformation-specific primary antibodies (Bax 6A7 [Sigma, no. B8429] and Bak NT [Millipore, no. 06536]). .. Species-specific Alexa fluor 647 or Cy5-conjugated secondary antibodies were used to detect activated Bax and Bak by flow cytometry.

    Activation Assay:

    Article Title: Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis
    Article Snippet: Paragraph title: Bax and Bak activation by cross-linking and flow cytometry ... For flow cytometry experiments, treated cells were fixed in 4% formalin for 15 min and stained with conformation-specific primary antibodies (Bax 6A7 [Sigma, no. B8429] and Bak NT [Millipore, no. 06536]).

    Protease Inhibitor:

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: The frozen hippocampi were homogenized with ice-cold lysis buffer that contained radio-immunoprecipitation assay (RIPA) buffer and protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA) , at a ratio of 1:10, for 1 hour, then centrifuged at 12000 g for 20 minutes at 4˚C. .. Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour.

    Cell Culture:

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: LN229 cells were cultured on 6-well plates and treated. .. The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Inhibition:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: .. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK). .. The membranes were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody.

    Imaging:

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich). .. Immunoblot bands were imaged by a gel imaging system (G: Box, Syngene, UK) and analysis of band intensity was performed with ImageJ software.

    Article Title: Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p, et al. Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p
    Article Snippet: Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl‐2 (SAB4500005, 1:1000) from Sigma‐Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C. .. Finally, the protein on the membrane was visualized using an image developing kit (P0020; Beyotime Biotechnology Co., Ltd.), and photographed using a Bio‐Rad gel imaging analysis system (Bio‐Rad).

    Protein Concentration:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: The protein concentration was measured by a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions. .. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK).

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: After determining the protein concentration with a Bio-Rad assay system (Bio-Rad, San Francisco, CA, USA), 100 μg aliquots of protein from each sample were denatured with sample buffer that consisted of 6.205 mM Tris-HCl, 10% glycerol, 2% sodium dodecyl sulphate (SDS), 0.01% bromophenol blue and 50 mM 2-mercaptoethanol (2-ME) (Sigma Aldrich, St. Louis, MO, USA) at 95˚C for 5 minutes and then separated using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 90 minutes at 120 voltage. .. Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour.

    Molecular Weight:

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour. .. The bands from various groups that corresponded to the appropriate molecular weight for each subunit were analyzed and values were compared using densitometric measurements by UVIdoc image analysis system (UVIdoc, Houston, TX, USA).

    Nucleic Acid Electrophoresis:

    Article Title: Psoralea glandulosa as a Potential Source of Anticancer Agents for Melanoma Treatment
    Article Snippet: Twenty micrograms of total protein, present in the supernatant, were loaded on each lane and separated by 4%–12% Novex Bis-Tris gel electrophoresis (NuPAGE, Invitrogen, Milan, Italy). .. Bcl-2 (SAB2500154, Sigma Aldrich, Milan, Italy) (1:500 dilution), Bax (B3428, Sigma-Aldrich) (1:2000 dilution), rabbit polyclonal anti-p53 (FL-393; sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:300 dilution) and α-tubulin (T5326; Sigma-Aldrich) (1:5000 dilution) antibodies were diluted in TBST and membranes incubated for 2 h at room temperature.

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: After determining the protein concentration with a Bio-Rad assay system (Bio-Rad, San Francisco, CA, USA), 100 μg aliquots of protein from each sample were denatured with sample buffer that consisted of 6.205 mM Tris-HCl, 10% glycerol, 2% sodium dodecyl sulphate (SDS), 0.01% bromophenol blue and 50 mM 2-mercaptoethanol (2-ME) (Sigma Aldrich, St. Louis, MO, USA) at 95˚C for 5 minutes and then separated using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 90 minutes at 120 voltage. .. Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour.

    Article Title: Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p, et al. Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p
    Article Snippet: Total protein in tissues and cells was extracted using RIPA cell lysis buffer (R0010; Solarbio) containing PMFS, which was then separated through sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). .. Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl‐2 (SAB4500005, 1:1000) from Sigma‐Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C.

    Magnetic Beads:

    Article Title: Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer
    Article Snippet: Immunocomplexes were captured using magnetic beads conjugated with protein A/G (Pierce, Waltham, MA) and then washed three times in lysis buffer. .. For immunoprecipitation, MCL-1 antibody (BD Biosciences, Cat # 554103) and conformation-specific antibodies for BAK (Millipore, Cat # 06-536) or BAX (Sigma, Cat # B8429) was used as primary antibody.

    Isolation:

    Article Title: Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis
    Article Snippet: The isolated mitochondria (30–60 µg) were incubated for 30 min in the dark at room temperature in 45 µL of cross-linking buffer (20 mM HEPES at pH 7.5, 250 mM sucrose, 1 mM EDTA, 50 mM KCl, 2.5 mM MgCl2 ) containing 0.5 mM BMH (1, 6–bismaleimidohexane; Thermo Scientific, no. 22331). .. For flow cytometry experiments, treated cells were fixed in 4% formalin for 15 min and stained with conformation-specific primary antibodies (Bax 6A7 [Sigma, no. B8429] and Bak NT [Millipore, no. 06536]).

    Transfection:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: Western blot analysis After 48 h of transfection with miR-455-3p mimics or inhibitors, the cells were lysed in lysis solution and prepared for protein extraction. .. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK).

    Article Title: Farnesylthiosalicylic acid sensitizes hepatocarcinoma cells to artemisinin derivatives
    Article Snippet: Mouse monoclonal anti-Bak antibody (Ab-2) and anti-Bax antibody (6A7) were purchased from Calbiochem (San Diego, CA, USA). .. Turbofect™ transfection regent was purchased from Thermo Fisher Scientific (Massachusetts, USA).

    Immunoprecipitation:

    Article Title: Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer
    Article Snippet: .. For immunoprecipitation, MCL-1 antibody (BD Biosciences, Cat # 554103) and conformation-specific antibodies for BAK (Millipore, Cat # 06-536) or BAX (Sigma, Cat # B8429) was used as primary antibody. .. For immunoblotting, primary antibodies included those against MCL-1 (Santa Cruz Biotechnology, Cat # sc-819) and tubulin (Sigma, T4026).

    Article Title: Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death
    Article Snippet: .. 500–1000 μ g protein was immunoprecipitated and incubated overnight at 4 °C with 2 μ g/ml mouse anti-BAK antibody (Ab-1; Merck Millipore, Billerica, MA, USA) or anti-BAX antibody (6A7, Sigma-Aldrich) and 10 μ l pan-mouse IgG Dynabeads (Life technologies, Inc.), washed with CHAPS lysis buffer and analyzed by western blotting using rabbit anti-BAK antibody (BD Bioscience) or anti-BAX antibody (Merck, Darmstadt, Germany). .. Immunoprecipitation of MCL-1 was performed in 500 μ l lysates containing up to 1000 μ g proteins, which were incubated overnight at 4 °C with 2 μ g/ml mouse anti-MCL-1 antibody (BD Biosciences) and 10 μ l pan-mouse IgG Dynabeads or Protein G Dynabeads (Life Technologies, Inc.) and washed with CHAPS buffer.

    Article Title: Recombinant VP1, an Akt Inhibitor, Suppresses Progression of Hepatocellular Carcinoma by Inducing Apoptosis and Modulation of CCL2 Production
    Article Snippet: .. Immunoprecipitation For detection of Ku70-Bax interactions in BNL and Hepa1-6 cells, cells treated with or without rVP1 were harvested in protein extraction reagents (Pierce), and 0.2–1 mg of cell lysates were immunoprecipitated with 4–10 µg of anti-Bax antibody (Sigma). .. The immunocomplexes were captured using an immunoprecipitation matrix (ExactaCruz C, Santa Cruz Biotechnology) following the manufacturer's protocol.

    Protein Extraction:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: Western blot analysis After 48 h of transfection with miR-455-3p mimics or inhibitors, the cells were lysed in lysis solution and prepared for protein extraction. .. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK).

    Article Title: Recombinant VP1, an Akt Inhibitor, Suppresses Progression of Hepatocellular Carcinoma by Inducing Apoptosis and Modulation of CCL2 Production
    Article Snippet: .. Immunoprecipitation For detection of Ku70-Bax interactions in BNL and Hepa1-6 cells, cells treated with or without rVP1 were harvested in protein extraction reagents (Pierce), and 0.2–1 mg of cell lysates were immunoprecipitated with 4–10 µg of anti-Bax antibody (Sigma). .. The immunocomplexes were captured using an immunoprecipitation matrix (ExactaCruz C, Santa Cruz Biotechnology) following the manufacturer's protocol.

    Lysis:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: Western blot analysis After 48 h of transfection with miR-455-3p mimics or inhibitors, the cells were lysed in lysis solution and prepared for protein extraction. .. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK).

    Article Title: Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer
    Article Snippet: Immunocomplexes were captured using magnetic beads conjugated with protein A/G (Pierce, Waltham, MA) and then washed three times in lysis buffer. .. For immunoprecipitation, MCL-1 antibody (BD Biosciences, Cat # 554103) and conformation-specific antibodies for BAK (Millipore, Cat # 06-536) or BAX (Sigma, Cat # B8429) was used as primary antibody.

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: The frozen hippocampi were homogenized with ice-cold lysis buffer that contained radio-immunoprecipitation assay (RIPA) buffer and protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA) , at a ratio of 1:10, for 1 hour, then centrifuged at 12000 g for 20 minutes at 4˚C. .. Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour.

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: Cells were harvested with RIPA lysis buffer (Pierce, USA) containing anti-protease cocktail (Roche, USA) and cleared by centrifugation at 4°C. .. The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Article Title: Targeting redox homeostasis in rhabdomyosarcoma cells: GSH-depleting agents enhance auranofin-induced cell death
    Article Snippet: .. 500–1000 μ g protein was immunoprecipitated and incubated overnight at 4 °C with 2 μ g/ml mouse anti-BAK antibody (Ab-1; Merck Millipore, Billerica, MA, USA) or anti-BAX antibody (6A7, Sigma-Aldrich) and 10 μ l pan-mouse IgG Dynabeads (Life technologies, Inc.), washed with CHAPS lysis buffer and analyzed by western blotting using rabbit anti-BAK antibody (BD Bioscience) or anti-BAX antibody (Merck, Darmstadt, Germany). .. Immunoprecipitation of MCL-1 was performed in 500 μ l lysates containing up to 1000 μ g proteins, which were incubated overnight at 4 °C with 2 μ g/ml mouse anti-MCL-1 antibody (BD Biosciences) and 10 μ l pan-mouse IgG Dynabeads or Protein G Dynabeads (Life Technologies, Inc.) and washed with CHAPS buffer.

    Article Title: Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p, et al. Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p
    Article Snippet: Total protein in tissues and cells was extracted using RIPA cell lysis buffer (R0010; Solarbio) containing PMFS, which was then separated through sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). .. Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl‐2 (SAB4500005, 1:1000) from Sigma‐Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C.

    Article Title: Safflor yellow B reduces hypoxia-mediated vasoconstriction by regulating endothelial micro ribonucleic acid/nitric oxide synthase signaling
    Article Snippet: Vascular endothelial cell or RAEC samples were homogenized with lysis buffer and centrifuged at 12,000 g for 15 min. .. After blocking with 1% bovine serum albumin for 120 min at 20 ± 2°C, the membranes were incubated with one of the following primary antibodies: anti-HIF-1α (1:1000, ab216842), anti-caspase 3 (1:500, ab32042), anti-eNOS (1:500, ab95254), anti-p-eNOSSer1177 (1:500, ab51038, all Abcam), anti-Bcl-2 (1:1000, SAB4500003), anti-Bax (1:1000, SAB4502546), anti-iNOS (1:1000, SAB4502011), anti-p-iNOSTyr151 (1:1000, SAB4301563), and anti-S100A1 (1:250, SAB4502708, all Sigma-Aldrich) overnight at 4°C prior to washing thrice (15-min each) with Tris-buffered saline containing Tween 20 (TBST).

    SDS Page:

    Article Title: Mutant BRAF upregulates MCL-1 to confer apoptosis resistance that is reversed by MCL-1 antagonism and cobimetinib in colorectal cancer
    Article Snippet: Immunoprecipitated proteins were eluted with 2x LDS sample buffer (Invitrogen, Cat # NP0008) and loaded onto a 14% SDS-PAGE gel for separation, followed by an electrical transfer onto PVDF membranes (Bio-Rad, Cat # 1620177). .. For immunoprecipitation, MCL-1 antibody (BD Biosciences, Cat # 554103) and conformation-specific antibodies for BAK (Millipore, Cat # 06-536) or BAX (Sigma, Cat # B8429) was used as primary antibody.

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: After determining the protein concentration with a Bio-Rad assay system (Bio-Rad, San Francisco, CA, USA), 100 μg aliquots of protein from each sample were denatured with sample buffer that consisted of 6.205 mM Tris-HCl, 10% glycerol, 2% sodium dodecyl sulphate (SDS), 0.01% bromophenol blue and 50 mM 2-mercaptoethanol (2-ME) (Sigma Aldrich, St. Louis, MO, USA) at 95˚C for 5 minutes and then separated using a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 90 minutes at 120 voltage. .. Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour.

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: Twenty to forty micrograms of each sample was subjected to 8 or 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad, USA). .. The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich).

    Article Title: Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p, et al. Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p
    Article Snippet: Total protein in tissues and cells was extracted using RIPA cell lysis buffer (R0010; Solarbio) containing PMFS, which was then separated through sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). .. Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl‐2 (SAB4500005, 1:1000) from Sigma‐Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C.

    Software:

    Article Title: Psoralea glandulosa as a Potential Source of Anticancer Agents for Melanoma Treatment
    Article Snippet: Bcl-2 (SAB2500154, Sigma Aldrich, Milan, Italy) (1:500 dilution), Bax (B3428, Sigma-Aldrich) (1:2000 dilution), rabbit polyclonal anti-p53 (FL-393; sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:300 dilution) and α-tubulin (T5326; Sigma-Aldrich) (1:5000 dilution) antibodies were diluted in TBST and membranes incubated for 2 h at room temperature. .. Bands were measured densitometrically with ImageJ software and their relative density calculated based on the density of the α-tubulin bands in each sample.

    Article Title: Involvement of Bax and Bcl-2 in Induction of Apoptosis by Essential Oils of Three Lebanese Salvia Species in Human Prostate Cancer Cells
    Article Snippet: Bcl-2 (SAB2500154, Sigma Aldrich) (1:500 dilution), -Bax (B3428, Sigma Aldrich) (1:2000 dilution), -cleaved caspase-9 (AB3629, Sigma Aldrich) (1:500 dilution), and α-tubulin (T5326; Sigma Aldrich) (1:5000 dilution) antibodies were diluted in Tris Buffered Saline, 0.1% Tween 20 (TBST)and membranes incubated for 2 h at room temperature. .. Bands were measured densitometrically by ImageJ software (NIH, Bethesda, MD; available at http://rsb.info.nih.gov/ij/ ) and their relative density calculated based on the density of the α-tubulin bands in each sample.

    Article Title: Activity of Metabotropic Glutamate Receptor 4 Suppresses Proliferation and Promotes Apoptosis With Inhibition of Gli-1 in Human Glioblastoma Cells
    Article Snippet: The primary antibodies and dilutions used in the experiments were as follows: rabbit anti-mGluR4 (1:1,000, Abcam); rabbit anti-Gli-1 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 3 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 8 polyclonal (1:1,000, Cell Signaling Technology); rabbit anti-caspase 9 polyclonal (1:1,000, Cell Signaling Technology); mouse anti-Bcl-2 monoclonal (1:1,000, Millipore); mouse anti-Bax monoclonal (1:1,000, Millipore); mouse anti-cyclin D1 monoclonal (1:1,000, Cell Signaling Technology); mouse anti-β-actin monoclonal (1:10,000, Sigma-Aldrich). .. Immunoblot bands were imaged by a gel imaging system (G: Box, Syngene, UK) and analysis of band intensity was performed with ImageJ software.

    Article Title: Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p, et al. Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p
    Article Snippet: Next, the membrane was probed with primary antibodies including mouse polyclonal antibodies to GAPDH (SAB1405848, 1 μg/mL) and NR1I3 (SAB1406904, 1 μg/mL), rabbit polyclonal antibodies to Bax (SAB4502546, 1:1000) and Bcl‐2 (SAB4500005, 1:1000) from Sigma‐Aldrich, and rabbit monoclonal antibody to proliferating cell nuclear antigen (PCNA; ab92552, 1:1000) from Abcam overnight at 4°C. .. The protein expression was analysed using the ImageJ software (National Institutes of Health).GAPDH was used as an internal control.

    Negative Control:

    Article Title: Recombinant VP1, an Akt Inhibitor, Suppresses Progression of Hepatocellular Carcinoma by Inducing Apoptosis and Modulation of CCL2 Production
    Article Snippet: Immunoprecipitation For detection of Ku70-Bax interactions in BNL and Hepa1-6 cells, cells treated with or without rVP1 were harvested in protein extraction reagents (Pierce), and 0.2–1 mg of cell lysates were immunoprecipitated with 4–10 µg of anti-Bax antibody (Sigma). .. Mouse IgG (Zymed, San Francisco, CA) was used as a negative control.

    Radio Immunoprecipitation:

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: The frozen hippocampi were homogenized with ice-cold lysis buffer that contained radio-immunoprecipitation assay (RIPA) buffer and protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA) , at a ratio of 1:10, for 1 hour, then centrifuged at 12000 g for 20 minutes at 4˚C. .. Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour.

    CCK-8 Assay:

    Article Title: Farnesylthiosalicylic acid sensitizes hepatocarcinoma cells to artemisinin derivatives
    Article Snippet: Mouse monoclonal anti-Bak antibody (Ab-2) and anti-Bax antibody (6A7) were purchased from Calbiochem (San Diego, CA, USA). .. Mouse monoclonal anti-Bak antibody (Ab-2) and anti-Bax antibody (6A7) were purchased from Calbiochem (San Diego, CA, USA).

    Concentration Assay:

    Article Title: MicroRNA-455-3p Inhibits Tumor Cell Proliferation and Induces Apoptosis in HCT116 Human Colon Cancer Cells
    Article Snippet: Soluble protein (the same concentration per lane) was resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to nitrocellulose membrane. .. Then the membranes were blocked with 5% non-fat dried milk and incubated with following primary antibodies overnight at 4o C: anti-p27 kinase inhibition protein (KIP) 1 antibody (P2092, Sigma-Aldrich), anti-p21 antibody (SAB4500065; Sigma-Aldrich), anti- B-cell lymphoma (Bcl)-2 antibody (AB1722, Sigma-Aldrich), anti-Bax antibody (B8429, Sigma-Aldrich), anti-pro-caspase 3 antibody (9662, Cell Signaling Technology), and anti-active caspase-3 antibody (ab2302, Abcam, Cambridge, UK).

    Staining:

    Article Title: Psoralea glandulosa as a Potential Source of Anticancer Agents for Melanoma Treatment
    Article Snippet: The transfer of proteins was verified by staining the nitrocellulose membranes with Ponceau S and the NovexBis-Tris gel with Brillant blue R. Membranes were blocked in Tris buffered saline containing 0.01% Tween-20 (TBST) and 5% non-fat dry milk at 4 °C overnight. .. Bcl-2 (SAB2500154, Sigma Aldrich, Milan, Italy) (1:500 dilution), Bax (B3428, Sigma-Aldrich) (1:2000 dilution), rabbit polyclonal anti-p53 (FL-393; sc-6243, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:300 dilution) and α-tubulin (T5326; Sigma-Aldrich) (1:5000 dilution) antibodies were diluted in TBST and membranes incubated for 2 h at room temperature.

    Article Title: Transforming Growth Factor-α Improves Memory Impairment and Neurogenesis Following Ischemia Reperfusion
    Article Snippet: .. Proteins were subsequently stained with anti-Bcl-2 and anti-Bax monoclonal antibodies (1:1000; Sigma Aldrich, MO, USA) for 2 hours followed by staining with alkaline phosphatase-conjugated secondary antibodies (1:10000, Sigma Aldrich, MO, USA) for 1 hour. .. Bands were detected using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl phosphate in the presence of nitroblue tetrazolium (Sigma Aldrich, St. Louis, MO, USA).

    Article Title: Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis
    Article Snippet: .. For flow cytometry experiments, treated cells were fixed in 4% formalin for 15 min and stained with conformation-specific primary antibodies (Bax 6A7 [Sigma, no. B8429] and Bak NT [Millipore, no. 06536]). .. Species-specific Alexa fluor 647 or Cy5-conjugated secondary antibodies were used to detect activated Bax and Bak by flow cytometry.

    Variant Assay:

    Article Title: Role of Calcium Signals on Hydrogen Peroxide-Induced Apoptosis in Human Myeloid HL-60 Cells
    Article Snippet: Materials HL-60 15-12 cell line (ECACC N° 88120805), a variant of HL-60 which is differentiating towards either neutrophils or monocytes was purchased from The European Collection of Cell Cultures (ECACC) (Dorset, U.K.). .. Dimethyl sulfoxide (DMSO), thapsigargin, N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (AC-DEVD-AMC), dithiothreitol (DTT) and anti-Bax monoclonal antibody (Clone 6A7) were obtained from Sigma (Madrid, Spain).

    other:

    Article Title: BAD sensitizes breast cancer cells to docetaxel with increased mitotic arrest and necroptosis
    Article Snippet: BAD, tubulin and BAX conformational antibody clone 6A7 were from Sigma-Aldrich.

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    Millipore mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh
    Mouse Monoclonal Anti Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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