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Santa Cruz Biotechnology anti gapdh
Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of <t>Fgf21,</t> Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to <t>Gapdh</t> and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P
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1) Product Images from "Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy"

Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy

Journal: The EMBO Journal

doi: 10.15252/embj.201899576

Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P
Figure Legend Snippet: Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

Techniques Used: Activation Assay, Mouse Assay

Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation UPR activation was analyzed by immunoblotting with indicated antibodies in GC muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p‐Eif2a, and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P
Figure Legend Snippet: Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation UPR activation was analyzed by immunoblotting with indicated antibodies in GC muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p‐Eif2a, and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

Techniques Used: Activation Assay, Mouse Assay

2) Product Images from "Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation"

Article Title: Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118473

S1P induces c-Src-dependent p42/p44 MAPK and p38 MAPK activation. HPAEpiCs were pretreated without or with PP1, AG1478, or AG1296 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of (A) phospho-p42/p44 MAPK, (B) phospho-p38 MAPK, or (C) phospho-JNK1/2 were determined by Western blot. Data are expressed as representatives of three independent experiments. (D) Cells were transfected with siRNA of scrambled, c-Src, EGFR, or PDGFR, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, and GAPDH proteins were determined by Western blot.
Figure Legend Snippet: S1P induces c-Src-dependent p42/p44 MAPK and p38 MAPK activation. HPAEpiCs were pretreated without or with PP1, AG1478, or AG1296 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of (A) phospho-p42/p44 MAPK, (B) phospho-p38 MAPK, or (C) phospho-JNK1/2 were determined by Western blot. Data are expressed as representatives of three independent experiments. (D) Cells were transfected with siRNA of scrambled, c-Src, EGFR, or PDGFR, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, and GAPDH proteins were determined by Western blot.

Techniques Used: Activation Assay, Incubation, Western Blot, Transfection

S1P stimulates c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3. (A) Cells were transfected with siRNA of scrambled, S1PR1, or S1PR3, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-c-Src, phospho-EGFR, phospho-PDGFR, phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, phospho-c-Jun, and GAPDH proteins were determined by Western blot. The figure represents one of three individual experiments (n = 3).
Figure Legend Snippet: S1P stimulates c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt- or JNK1/2-dependent AP-1 activation are mediated via S1PR1/3. (A) Cells were transfected with siRNA of scrambled, S1PR1, or S1PR3, and then incubated with S1P (10 μM) for the indicated time intervals. The levels of phospho-c-Src, phospho-EGFR, phospho-PDGFR, phospho-p38 MAPK, phospho-p42/p44 MAPK, phospho-JNK1/2, phospho-c-Jun, and GAPDH proteins were determined by Western blot. The figure represents one of three individual experiments (n = 3).

Techniques Used: Activation Assay, Transfection, Incubation, Western Blot

3) Product Images from "Regional Gene Repression by DNA Double-Strand Breaks in G1 Phase Cells"

Article Title: Regional Gene Repression by DNA Double-Strand Breaks in G1 Phase Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00181-19

Persistent DSBs impede ongoing and induced transcription of an endogenous gene. (A) RT-qPCR analysis of total transcript levels of Ifit1 (primer pair P2) in G 1 -arrested LigIV −/− cells at the indicated time points after treatment with 100 U/ml IFN-β. The transcript levels relative to the levels in untreated cells (0-h time point) are shown. (B) Immunoblot (IB) analysis of γ-H2AX levels in G 1 -arrested LigIV −/− cells that were untreated or treated with 100 U/ml IFN-β or 5 μg/ml of the DNA-damaging agent bleomycin (Bleocin) for 24 h. GAPDH expression is shown as a protein loading control. (C) Schematic of the Ifit1 locus. gRNA target sites are denoted by yellow arrows, and the qPCR primers used to detect Ifit1 transcripts are shown as red arrows. The distances between gRNA target sites and the Ifit1 promoter are indicated. (D) Schematic of the Southern blotting strategy for detecting cleaved alleles at the Ifit1 intronic gRNA target site (g Ifit1 intron) (top) and Southern blots showing intact and cut Ifit1 alleles at 24 h (left) or 28 h (right) after nucleofection of G 1 -arrested, LigIV −/− : iCas9 cells with an empty gRNA vector (gEmpty) or the g Ifit1 intron (bottom). Cells were treated with 100 U/ml IFN-β, as described in the work flow presented in panel F (left) or as described in the work flow presented in panel I (right). (E) Schematic of the Southern blotting strategy for detecting cleaved alleles at the Ifit1 3′ gRNA target site (g Ifit1 3′ ) (top) and Southern blots showing intact and cut Ifit1 alleles, as described in the legend to panel D (bottom). (F) Work flow for the ongoing transcription experiments whose results are shown in panels G and H. (G) RT-qPCR analysis of nascent transcript levels of Ifit1 (primer pairs P1, P2, and P3) and a control interferon-stimulated gene on a separate chromosome, Isg15 , at 24 h after nucleofection with a gRNA targeting the Ifit1 intron (g Ifit1 intron). Cells were treated with 100 U/ml IFN-β 2 h prior to nucleofection and pulsed with EU 1 h prior to harvesting for RNA isolation. Nascent transcripts were isolated with the Click-iT nascent RNA capture technology. Transcript levels are shown relative to the levels in cells nucleofected with an empty gRNA vector. (H) RT-qPCR analysis of nascent transcript levels of Ifit1 , as described in the legend to panel G, after nucleofection with a gRNA targeting the region ∼3 kb downstream of the Ifit1 gene body (g Ifit1 3′). (I) Work flow for the induced transcription experiments whose results are shown in panels J to M. (J) RT-qPCR analysis of induced transcript levels of Ifit1 (primer pairs P1, P2, and P3) and Isg15 at 28 h after nucleofection with a gRNA targeting the Ifit1 intron ( gIfit1 intron). Cells were treated with 100 U/ml IFN-β for 4 h at 24 h after nucleofection. Transcript levels are shown relative to the levels in cells nucleofected with gEmpty. (K) RT-qPCR analysis of induced transcript levels of Ifit1 , as described in the legend to panel J, after nucleofection with g Ifit1 3′. (L) RT-qPCR analysis of induced transcript levels of Ifit1 and Isg15 in cells arrested in G 1 with palbociclib, followed by nucleofection and treatment, as described in the legend to panel J. (M) RT-qPCR analysis of induced transcript levels of Ifit1 and Isg15 in cells arrested in G 1 with palbociclib, followed by nucleofection and treatment, as described in the legend to panel K. Data from panels G, H, and J to M represent those from 3 independent experiments ( n = 3). Error bars show the SEM. *, P
Figure Legend Snippet: Persistent DSBs impede ongoing and induced transcription of an endogenous gene. (A) RT-qPCR analysis of total transcript levels of Ifit1 (primer pair P2) in G 1 -arrested LigIV −/− cells at the indicated time points after treatment with 100 U/ml IFN-β. The transcript levels relative to the levels in untreated cells (0-h time point) are shown. (B) Immunoblot (IB) analysis of γ-H2AX levels in G 1 -arrested LigIV −/− cells that were untreated or treated with 100 U/ml IFN-β or 5 μg/ml of the DNA-damaging agent bleomycin (Bleocin) for 24 h. GAPDH expression is shown as a protein loading control. (C) Schematic of the Ifit1 locus. gRNA target sites are denoted by yellow arrows, and the qPCR primers used to detect Ifit1 transcripts are shown as red arrows. The distances between gRNA target sites and the Ifit1 promoter are indicated. (D) Schematic of the Southern blotting strategy for detecting cleaved alleles at the Ifit1 intronic gRNA target site (g Ifit1 intron) (top) and Southern blots showing intact and cut Ifit1 alleles at 24 h (left) or 28 h (right) after nucleofection of G 1 -arrested, LigIV −/− : iCas9 cells with an empty gRNA vector (gEmpty) or the g Ifit1 intron (bottom). Cells were treated with 100 U/ml IFN-β, as described in the work flow presented in panel F (left) or as described in the work flow presented in panel I (right). (E) Schematic of the Southern blotting strategy for detecting cleaved alleles at the Ifit1 3′ gRNA target site (g Ifit1 3′ ) (top) and Southern blots showing intact and cut Ifit1 alleles, as described in the legend to panel D (bottom). (F) Work flow for the ongoing transcription experiments whose results are shown in panels G and H. (G) RT-qPCR analysis of nascent transcript levels of Ifit1 (primer pairs P1, P2, and P3) and a control interferon-stimulated gene on a separate chromosome, Isg15 , at 24 h after nucleofection with a gRNA targeting the Ifit1 intron (g Ifit1 intron). Cells were treated with 100 U/ml IFN-β 2 h prior to nucleofection and pulsed with EU 1 h prior to harvesting for RNA isolation. Nascent transcripts were isolated with the Click-iT nascent RNA capture technology. Transcript levels are shown relative to the levels in cells nucleofected with an empty gRNA vector. (H) RT-qPCR analysis of nascent transcript levels of Ifit1 , as described in the legend to panel G, after nucleofection with a gRNA targeting the region ∼3 kb downstream of the Ifit1 gene body (g Ifit1 3′). (I) Work flow for the induced transcription experiments whose results are shown in panels J to M. (J) RT-qPCR analysis of induced transcript levels of Ifit1 (primer pairs P1, P2, and P3) and Isg15 at 28 h after nucleofection with a gRNA targeting the Ifit1 intron ( gIfit1 intron). Cells were treated with 100 U/ml IFN-β for 4 h at 24 h after nucleofection. Transcript levels are shown relative to the levels in cells nucleofected with gEmpty. (K) RT-qPCR analysis of induced transcript levels of Ifit1 , as described in the legend to panel J, after nucleofection with g Ifit1 3′. (L) RT-qPCR analysis of induced transcript levels of Ifit1 and Isg15 in cells arrested in G 1 with palbociclib, followed by nucleofection and treatment, as described in the legend to panel J. (M) RT-qPCR analysis of induced transcript levels of Ifit1 and Isg15 in cells arrested in G 1 with palbociclib, followed by nucleofection and treatment, as described in the legend to panel K. Data from panels G, H, and J to M represent those from 3 independent experiments ( n = 3). Error bars show the SEM. *, P

Techniques Used: Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Southern Blot, Plasmid Preparation, Isolation

4) Product Images from "Capsaicin triggers immunogenic PEL cell death, stimulates DCs and reverts PEL-induced immune suppression"

Article Title: Capsaicin triggers immunogenic PEL cell death, stimulates DCs and reverts PEL-induced immune suppression

Journal: Oncotarget

doi:

The reduction of STAT3 phosphorylation is involved in Capsaicin-mediated cell death A. BC3 and BCBL1 were treated for 24 hours with Capaicin (200 μM) and STAT3 tyrosine phosphorylation was evaluated by western blot analysis. Total STAT3 and β-Actin were included as control. A representative experiment is shown and the mean plus SD of the densitometric analysis of the specific proteins on β-Actin of three independent experiments is also reported; B. BC3 and BCBL1 were treated for 24 hours with AG490 (100 μM) and STAT3 tyrosine phosphorylation was evaluated by western blot analysis. Total STAT3 and Actin were included as control. A representative experiment is shown and the mean plus SD of the densitometric analysis of the specific proteins on β-Actin of three independent experiments is also reported; C. BCBL1 PEL cells were treated for 24 hours with Capsaicin (200 μM) in the presence or in the absence of orthovanadate (OV) (100 μM) and STAT3 tyrosine phosphorylation was evaluated by western blot analysis. Total STAT3 and GAPDH were included as control. Mean plus SD of the densitometric analysis of the specific proteins on GAPDH of three independent experiments is also reported; D. Cell viability of BCBL1 PEL cells treated for 24 hours with Capaicin (200 μM) in the presence or in the absence of OV (100 μM). Cells were counted by trypan blue exclusion and mean of the percentage of cell survival plus SD of three independent experiments is shown.
Figure Legend Snippet: The reduction of STAT3 phosphorylation is involved in Capsaicin-mediated cell death A. BC3 and BCBL1 were treated for 24 hours with Capaicin (200 μM) and STAT3 tyrosine phosphorylation was evaluated by western blot analysis. Total STAT3 and β-Actin were included as control. A representative experiment is shown and the mean plus SD of the densitometric analysis of the specific proteins on β-Actin of three independent experiments is also reported; B. BC3 and BCBL1 were treated for 24 hours with AG490 (100 μM) and STAT3 tyrosine phosphorylation was evaluated by western blot analysis. Total STAT3 and Actin were included as control. A representative experiment is shown and the mean plus SD of the densitometric analysis of the specific proteins on β-Actin of three independent experiments is also reported; C. BCBL1 PEL cells were treated for 24 hours with Capsaicin (200 μM) in the presence or in the absence of orthovanadate (OV) (100 μM) and STAT3 tyrosine phosphorylation was evaluated by western blot analysis. Total STAT3 and GAPDH were included as control. Mean plus SD of the densitometric analysis of the specific proteins on GAPDH of three independent experiments is also reported; D. Cell viability of BCBL1 PEL cells treated for 24 hours with Capaicin (200 μM) in the presence or in the absence of OV (100 μM). Cells were counted by trypan blue exclusion and mean of the percentage of cell survival plus SD of three independent experiments is shown.

Techniques Used: Western Blot

5) Product Images from "Wnt-related SynGAP1 is a neuroprotective factor of glutamatergic synapses against Aβ oligomers"

Article Title: Wnt-related SynGAP1 is a neuroprotective factor of glutamatergic synapses against Aβ oligomers

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2015.00227

Wnt-5a increases the phosphorylation levels of SynGAP by a mechanism dependent of CamKII. (A) WB analysis of pSynGAP (S1123) and p-calmodulin-dependent protein kinase II (CaMKII) levels in hippocampal neurons treated with recombinant Wnt-5a (300 ng/mL) at different time points. (B) Densitometric analysis of pSynGAP1, shown in (A) . (C) Densitometric analysis of pCaMKII, shown in (A) . The results are presented as the mean of n = 3 experiments and were normalized to GAPDH expression. (D) Representative neurite images of SynGAP immunofluorescence (green) from samples subjected to rWnt-5a (300 ng/mL) treatment for different time points, white bar represents 5 μm. (E) Quantification of the cluster density of SynGAP (cluster number/μm) in neurons described in (A) . (F) Model of the effect of Wnt-5a on SynGAP function. After binding of Wnt-5a to their receptors, there is an increase of intracellular calcium which could be due to a release from internal stores (Wnt/Ca +2 pathway) or modulation of NMDAR. Activation of CaMKII induces the increase in phosphorylation levels of SynGAP at S1123 and migration from dendritic clusters to a diffuse pool in the dendritic shaft. Statistical analysis was performed using one-way ANOVA, followed by Dunn’s Multiple Comparison Test. ∗ p
Figure Legend Snippet: Wnt-5a increases the phosphorylation levels of SynGAP by a mechanism dependent of CamKII. (A) WB analysis of pSynGAP (S1123) and p-calmodulin-dependent protein kinase II (CaMKII) levels in hippocampal neurons treated with recombinant Wnt-5a (300 ng/mL) at different time points. (B) Densitometric analysis of pSynGAP1, shown in (A) . (C) Densitometric analysis of pCaMKII, shown in (A) . The results are presented as the mean of n = 3 experiments and were normalized to GAPDH expression. (D) Representative neurite images of SynGAP immunofluorescence (green) from samples subjected to rWnt-5a (300 ng/mL) treatment for different time points, white bar represents 5 μm. (E) Quantification of the cluster density of SynGAP (cluster number/μm) in neurons described in (A) . (F) Model of the effect of Wnt-5a on SynGAP function. After binding of Wnt-5a to their receptors, there is an increase of intracellular calcium which could be due to a release from internal stores (Wnt/Ca +2 pathway) or modulation of NMDAR. Activation of CaMKII induces the increase in phosphorylation levels of SynGAP at S1123 and migration from dendritic clusters to a diffuse pool in the dendritic shaft. Statistical analysis was performed using one-way ANOVA, followed by Dunn’s Multiple Comparison Test. ∗ p

Techniques Used: Western Blot, Recombinant, Expressing, Immunofluorescence, Binding Assay, Activation Assay, Migration

6) Product Images from "SMaRT lncRNA controls translation of a G‐quadruplex‐containing mRNA antagonizing the DHX36 helicase"

Article Title: SMaRT lncRNA controls translation of a G‐quadruplex‐containing mRNA antagonizing the DHX36 helicase

Journal: EMBO Reports

doi: 10.15252/embr.201949942

Lnc‐SMaRT molecular interactome Localization on the lnc‐SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc‐SMaRT pull‐down experiment. Left panel: qPCR analysis of lnc‐SMaRT enrichment in the RNA pull‐down performed in C2C12 cells at day 2 of differentiation (D2); Set#1 and Set#2 probes were used against lnc‐SMaRT together with a control set of probes against LacZ mRNA (LacZ). Data are expressed in percentage of input and presented as the mean ± s.e.m. of three biological replicates (dots). Right panel: Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc‐SMaRT pull‐down; GAPDH was used as negative control. Representative results from three independent experiments are shown. Upper panel: Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation. Input sample (IN) accounts for 2.5% of the extract. Representative results from three independent experiments are shown. Lower panel: qPCR analysis of lnc‐SMaRT enrichment in DHX36 RIP‐derived RNA extracts. WBP4 was used as positive control 31 , while Neat1 (a lncRNA expressed at comparable level of lnc‐SMaRT according to the RNA sequencing) and Rps7 were used as negative controls. Data are expressed as percentage of input normalized on IgG control and presented as the mean ± s.e.m. of three biological replicates (dots). RT–PCR validation of Mlx‐α, Mlx‐β, and Mlx‐γ mRNA enrichment upon the lnc‐SMaRT pull‐down performed with Set#1 and Set#2 probes; a control set of probes against LacZ mRNA (LacZ) was used as negative control. GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown. RT–PCR analysis of Mlx‐α, Mlx‐β, and Mlx‐γ mRNA enrichment in DHX36 RIP‐derived RNA extracts in samples treated with control siRNA (si‐SCR, upper panel) or siRNA against lnc‐SMaRT (si‐SMaRT, lower panel). GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown. Upper panel: Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG‐tagged MLX (MLX‐α, MLX‐β, and MLX‐γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE). FLAG tag has been inserted at the N‐terminus of MLX protein isoforms. HPRT was used as loading control. Representative results from three independent experiments are shown. Middle panel: Quantification of FLAG‐tagged protein levels normalized on HPRT signals. Data are expressed as the mean ± s.d. of three biological replicates (dots). Statistical analysis was performed with two‐way ANOVA followed by Bonferroni's multiple comparisons test. *** P
Figure Legend Snippet: Lnc‐SMaRT molecular interactome Localization on the lnc‐SMaRT transcript of the two sets of biotinylated probes (Set#1 and Set#2) used for lnc‐SMaRT pull‐down experiment. Left panel: qPCR analysis of lnc‐SMaRT enrichment in the RNA pull‐down performed in C2C12 cells at day 2 of differentiation (D2); Set#1 and Set#2 probes were used against lnc‐SMaRT together with a control set of probes against LacZ mRNA (LacZ). Data are expressed in percentage of input and presented as the mean ± s.e.m. of three biological replicates (dots). Right panel: Western blot analysis showing the specific enrichment of the DHX36 helicase in lnc‐SMaRT pull‐down; GAPDH was used as negative control. Representative results from three independent experiments are shown. Upper panel: Western blot with DHX36 antibodies on protein extracts from DHX36 RNA immunoprecipitation. Input sample (IN) accounts for 2.5% of the extract. Representative results from three independent experiments are shown. Lower panel: qPCR analysis of lnc‐SMaRT enrichment in DHX36 RIP‐derived RNA extracts. WBP4 was used as positive control 31 , while Neat1 (a lncRNA expressed at comparable level of lnc‐SMaRT according to the RNA sequencing) and Rps7 were used as negative controls. Data are expressed as percentage of input normalized on IgG control and presented as the mean ± s.e.m. of three biological replicates (dots). RT–PCR validation of Mlx‐α, Mlx‐β, and Mlx‐γ mRNA enrichment upon the lnc‐SMaRT pull‐down performed with Set#1 and Set#2 probes; a control set of probes against LacZ mRNA (LacZ) was used as negative control. GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown. RT–PCR analysis of Mlx‐α, Mlx‐β, and Mlx‐γ mRNA enrichment in DHX36 RIP‐derived RNA extracts in samples treated with control siRNA (si‐SCR, upper panel) or siRNA against lnc‐SMaRT (si‐SMaRT, lower panel). GAPDH was used as negative control. Input sample (IN) accounts for 10% of the extract. Representative results from three independent experiments are shown. Upper panel: Western blot with FLAG antibody on protein extracts from HeLa cells overexpressing the indicated isoforms of FLAG‐tagged MLX (MLX‐α, MLX‐β, and MLX‐γ) in control condition (CTRL) or in overexpression of lncSMaRT (OE). FLAG tag has been inserted at the N‐terminus of MLX protein isoforms. HPRT was used as loading control. Representative results from three independent experiments are shown. Middle panel: Quantification of FLAG‐tagged protein levels normalized on HPRT signals. Data are expressed as the mean ± s.d. of three biological replicates (dots). Statistical analysis was performed with two‐way ANOVA followed by Bonferroni's multiple comparisons test. *** P

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Negative Control, Immunoprecipitation, Derivative Assay, Positive Control, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Over Expression, FLAG-tag

Lnc‐SMaRT depletion inhibits myoblast differentiation qPCR analysis of MyoD and Myog RNA expression in C2C12 cells undergoing differentiation at the indicated time points. Data are presented as the mean ± s.e.m. of three biological replicates (dots) normalized against the GAPDH mRNA. RT–PCR showing the expression levels of lnc‐SMaRT in RNA extracted from the indicated muscle tissues of control (WT) and mdx mice ( mdx ). GAPDH was used as control. Representative results from three independent experiments are shown. RT–PCR on nuclear (N) and cytoplasmic (C) extracts showing the subcellular levels of lnc‐SMaRT RNA upon siRNAs treatment. RNA was isolated from C2C12 myoblasts treated with either control siRNA (si‐SCR) or siRNA against lnc‐SMaRT (si‐SMaRT‐1) and induced to differentiate for 2 days. GAPDH mRNA and pre‐mRNA (pre‐GAPDH) were used, respectively, as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown. qPCR analysis of indicated muscle differentiation marker (MyoD, Myog, Mef2C, Mck, and Dys) mRNA expression in C2C12 cells undergoing differentiation (D2) in control samples (black bars) or samples depleted of lnc‐SMaRT with two different siRNAs (gray and white bars). The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to the si‐SCR sample set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. * P
Figure Legend Snippet: Lnc‐SMaRT depletion inhibits myoblast differentiation qPCR analysis of MyoD and Myog RNA expression in C2C12 cells undergoing differentiation at the indicated time points. Data are presented as the mean ± s.e.m. of three biological replicates (dots) normalized against the GAPDH mRNA. RT–PCR showing the expression levels of lnc‐SMaRT in RNA extracted from the indicated muscle tissues of control (WT) and mdx mice ( mdx ). GAPDH was used as control. Representative results from three independent experiments are shown. RT–PCR on nuclear (N) and cytoplasmic (C) extracts showing the subcellular levels of lnc‐SMaRT RNA upon siRNAs treatment. RNA was isolated from C2C12 myoblasts treated with either control siRNA (si‐SCR) or siRNA against lnc‐SMaRT (si‐SMaRT‐1) and induced to differentiate for 2 days. GAPDH mRNA and pre‐mRNA (pre‐GAPDH) were used, respectively, as cytoplasmic and nuclear controls. Representative results from three independent experiments are shown. qPCR analysis of indicated muscle differentiation marker (MyoD, Myog, Mef2C, Mck, and Dys) mRNA expression in C2C12 cells undergoing differentiation (D2) in control samples (black bars) or samples depleted of lnc‐SMaRT with two different siRNAs (gray and white bars). The RNA expression levels in qPCR analyses were normalized against GAPDH mRNA and expressed as relative quantities with respect to the si‐SCR sample set to a value of 1. Data are presented as the mean ± s.e.m. of three biological replicates (dots). Statistical analysis was performed with analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. * P

Techniques Used: Real-time Polymerase Chain Reaction, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay, Isolation, Marker

7) Product Images from "MicroRNA 1253 regulation of WAVE2 and its relevance to health disparities in hypertension"

Article Title: MicroRNA 1253 regulation of WAVE2 and its relevance to health disparities in hypertension

Journal: bioRxiv

doi: 10.1101/833673

Overexpression of miR-1253 in HAECs and HUVECs reduces expression of WAVE2. 50 nM miR-1253 was transfected into HAECs (n=3) (A) and HUVECs (n=5) (B) for 48 hrs and over-expressed in each cell line compared with a scrambled negative control mimic (scr.; top left). WAVE2 expression was normalized to GAPDH in each cell line and shown relative to scrambled (scr.; top right). WAVE2 proteins levels were normalized to Beta Actin (HAECs) or GAPDH (HUVECs) and shown relative to a scrambled control (scr.; bottom left). Representative immunoblots are shown for WAVE2 and loading controls in each cell line (bottom right) * P
Figure Legend Snippet: Overexpression of miR-1253 in HAECs and HUVECs reduces expression of WAVE2. 50 nM miR-1253 was transfected into HAECs (n=3) (A) and HUVECs (n=5) (B) for 48 hrs and over-expressed in each cell line compared with a scrambled negative control mimic (scr.; top left). WAVE2 expression was normalized to GAPDH in each cell line and shown relative to scrambled (scr.; top right). WAVE2 proteins levels were normalized to Beta Actin (HAECs) or GAPDH (HUVECs) and shown relative to a scrambled control (scr.; bottom left). Representative immunoblots are shown for WAVE2 and loading controls in each cell line (bottom right) * P

Techniques Used: Over Expression, Expressing, Transfection, Negative Control, Western Blot

8) Product Images from "Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization"

Article Title: Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19020605

Detection of the NanoLuc reporter activity by endogenous NFAT in HEK293 cells depending on Ca 2+ -influx through CRAC channels. ( A ) HEK293 cells stably expressing human STIM1 and Orai1 (HEK293 S1/O1) were established by the retrovirus vector system as described in the Materials and Methods. Total lysates of the parental HEK293 and S1/O1 cells were analyzed by Western blotting (WB) with antibodies against STIM1, Orai1, STIM2, ALG-2, and GAPDH (used as a loading control), respectively. Multiple bands indicated by half-square brackets a and b probably reflect differences in glycosylated forms of Orai1. ( B ) One day after transfection with the 9×IL8 NFAT-RE NanoLuc reporter plasmid and Fluc plasmid, the parental HEK293 and S1/O1 cells were pre-treated with or without FK506 (10 μM) or CRAC channel inhibitor BTP2 (1 μM; vehicle, 0.1% DMSO) for 1 h, and then stimulated with thapsigargin (TG, 200 nM; vehicle, 0.02% DMSO) for 6 h, followed by luciferase assays. A NanoLuc reporter containing no NFAT-RE (no RE) was used as a negative control. Dots and bars represent individual and averaged values obtained from triplicate assays, respectively. The presence and absence of the reagents are indicated by + and −, respectively.
Figure Legend Snippet: Detection of the NanoLuc reporter activity by endogenous NFAT in HEK293 cells depending on Ca 2+ -influx through CRAC channels. ( A ) HEK293 cells stably expressing human STIM1 and Orai1 (HEK293 S1/O1) were established by the retrovirus vector system as described in the Materials and Methods. Total lysates of the parental HEK293 and S1/O1 cells were analyzed by Western blotting (WB) with antibodies against STIM1, Orai1, STIM2, ALG-2, and GAPDH (used as a loading control), respectively. Multiple bands indicated by half-square brackets a and b probably reflect differences in glycosylated forms of Orai1. ( B ) One day after transfection with the 9×IL8 NFAT-RE NanoLuc reporter plasmid and Fluc plasmid, the parental HEK293 and S1/O1 cells were pre-treated with or without FK506 (10 μM) or CRAC channel inhibitor BTP2 (1 μM; vehicle, 0.1% DMSO) for 1 h, and then stimulated with thapsigargin (TG, 200 nM; vehicle, 0.02% DMSO) for 6 h, followed by luciferase assays. A NanoLuc reporter containing no NFAT-RE (no RE) was used as a negative control. Dots and bars represent individual and averaged values obtained from triplicate assays, respectively. The presence and absence of the reagents are indicated by + and −, respectively.

Techniques Used: Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Transfection, Luciferase, Negative Control

9) Product Images from "ErbB3 Phosphorylation as Central Event in Adaptive Resistance to Targeted Therapy in Metastatic Melanoma: Early Detection in CTCs during Therapy and Insights into Regulation by Autocrine Neuregulin"

Article Title: ErbB3 Phosphorylation as Central Event in Adaptive Resistance to Targeted Therapy in Metastatic Melanoma: Early Detection in CTCs during Therapy and Insights into Regulation by Autocrine Neuregulin

Journal: Cancers

doi: 10.3390/cancers11101425

Different kinetics of ErbB3 activation occur in BRAF-mutated melanoma cell lines. ( A ) WM266 (left panel), WM115 (middle panel) and LOX IMVI (right panel) melanoma cells were starved and then treated with BRAFi (0.5 μM) for different times (from 2 h to 72 h). ErbB3/Akt axis is early activated after BRAF inhibitor treatments with a different manner. Densitometric analysis values were used to evaluate pErbB3/ErbB3, pERK/ERK and pAkt/Akt expression with respect to the control untreated cells. ( B ) In the same experimantal condition (panel A) also total RNA was extracted and subjected to qRT-PCR in order to evaluate the kinetic activation of of NRG1, ErbB3 and FOXD3 genes. Results are represented as a “heat map”. Colors in the heat map indicate no increase in gene expression (green); moderate increase (yellow) or a strong increase (red), respectively. GAPDH reference gene was used for normalization.
Figure Legend Snippet: Different kinetics of ErbB3 activation occur in BRAF-mutated melanoma cell lines. ( A ) WM266 (left panel), WM115 (middle panel) and LOX IMVI (right panel) melanoma cells were starved and then treated with BRAFi (0.5 μM) for different times (from 2 h to 72 h). ErbB3/Akt axis is early activated after BRAF inhibitor treatments with a different manner. Densitometric analysis values were used to evaluate pErbB3/ErbB3, pERK/ERK and pAkt/Akt expression with respect to the control untreated cells. ( B ) In the same experimantal condition (panel A) also total RNA was extracted and subjected to qRT-PCR in order to evaluate the kinetic activation of of NRG1, ErbB3 and FOXD3 genes. Results are represented as a “heat map”. Colors in the heat map indicate no increase in gene expression (green); moderate increase (yellow) or a strong increase (red), respectively. GAPDH reference gene was used for normalization.

Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR

WM266 and WM115 melanoma cells show different mechanisms of NRG-1 activation. ( A ) WM266 and WM115 cells were transfected with NRG1-reporter plasmid and treated or not with BRAF inhibitor to 0.5 µM for 24 h. The luciferase gene was cloned downstream of NRG1. The luciferase activity was evaluated by the Renilla Luciferase assay. The results show that NRG1 gene promoter activity is enhanced after BRAF inhibitor treatment only in WM115 cells while no luciferase induction occurred in WM266 cell line. ( B ) Melanoma cells were co-treated with BRAF inhibitor (0.5 µM) and actinomicin D transcription inhibitor (5 µg/mL) for 24 h and subsequently collected and subjected to qRT-PCR analysis to evaluate ErbB3 and NRG1 gene expression. Results show that only in WM115 cells NRG1 expression is under transcriptional control. GAPDH reference gene was used for normalization. *: p
Figure Legend Snippet: WM266 and WM115 melanoma cells show different mechanisms of NRG-1 activation. ( A ) WM266 and WM115 cells were transfected with NRG1-reporter plasmid and treated or not with BRAF inhibitor to 0.5 µM for 24 h. The luciferase gene was cloned downstream of NRG1. The luciferase activity was evaluated by the Renilla Luciferase assay. The results show that NRG1 gene promoter activity is enhanced after BRAF inhibitor treatment only in WM115 cells while no luciferase induction occurred in WM266 cell line. ( B ) Melanoma cells were co-treated with BRAF inhibitor (0.5 µM) and actinomicin D transcription inhibitor (5 µg/mL) for 24 h and subsequently collected and subjected to qRT-PCR analysis to evaluate ErbB3 and NRG1 gene expression. Results show that only in WM115 cells NRG1 expression is under transcriptional control. GAPDH reference gene was used for normalization. *: p

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Clone Assay, Activity Assay, Quantitative RT-PCR, Expressing

10) Product Images from "Overexpression of Napsin A resensitizes drug-resistant lung cancer A549 cells to gefitinib by inhibiting EMT"

Article Title: Overexpression of Napsin A resensitizes drug-resistant lung cancer A549 cells to gefitinib by inhibiting EMT

Journal: Oncology Letters

doi: 10.3892/ol.2018.8963

Characterization of A549-GFT and A549 cells. (A) Cells were maintained in 96-well plates and cell proliferation at indicated time intervals was assessed by MTT assay. Growth curves of the two cells were drawn. (B) Cells were incubated in 100 µl medium for 24 h, and then were treated with 0, 1, 3, 5, 7, 9 and 18 µmol/l gefitinib for 48 h. Cell proliferation was evaluated by MTT assay. The IC 50 values of the two cell lines were calculated. (C) A549-GFT and A549 cells were exposed to 2 µmol/l gefitinib for 48 h, and then cell morphology was visualized by microscopy. (D) E-cadherin and Vimentin expression in A549-GFT and A549 cells were assessed by western blot analysis. GAPDH was detected as an internal standard. *P
Figure Legend Snippet: Characterization of A549-GFT and A549 cells. (A) Cells were maintained in 96-well plates and cell proliferation at indicated time intervals was assessed by MTT assay. Growth curves of the two cells were drawn. (B) Cells were incubated in 100 µl medium for 24 h, and then were treated with 0, 1, 3, 5, 7, 9 and 18 µmol/l gefitinib for 48 h. Cell proliferation was evaluated by MTT assay. The IC 50 values of the two cell lines were calculated. (C) A549-GFT and A549 cells were exposed to 2 µmol/l gefitinib for 48 h, and then cell morphology was visualized by microscopy. (D) E-cadherin and Vimentin expression in A549-GFT and A549 cells were assessed by western blot analysis. GAPDH was detected as an internal standard. *P

Techniques Used: MTT Assay, Incubation, Microscopy, Expressing, Western Blot

Effect of Napsin A overexpression on gefitinib-induced apoptosis in A549-GFT cells. Napsin A was overexpressed in A549 and A549-GFT cells by PLJM1-Napsin A lentivirus infection, and the cells were then exposed to 2 µmol/l gefitinib for 48 h. (A) Apoptosis of A549 and A549-GFT cells was evaluated by Annexin V/propidium iodide staining assay and flow cytometry. (B) Western blot analysis was used to detect the protein expression levels of E-cadherin, Vimentin, p-FAK and total FAK. GAPDH was detected as an internal standard. **P
Figure Legend Snippet: Effect of Napsin A overexpression on gefitinib-induced apoptosis in A549-GFT cells. Napsin A was overexpressed in A549 and A549-GFT cells by PLJM1-Napsin A lentivirus infection, and the cells were then exposed to 2 µmol/l gefitinib for 48 h. (A) Apoptosis of A549 and A549-GFT cells was evaluated by Annexin V/propidium iodide staining assay and flow cytometry. (B) Western blot analysis was used to detect the protein expression levels of E-cadherin, Vimentin, p-FAK and total FAK. GAPDH was detected as an internal standard. **P

Techniques Used: Over Expression, Infection, Staining, Flow Cytometry, Cytometry, Western Blot, Expressing

11) Product Images from "Interaction of the Lysophospholipase PNPLA7 with Lipid Droplets through the Catalytic Region"

Article Title: Interaction of the Lysophospholipase PNPLA7 with Lipid Droplets through the Catalytic Region

Journal: Molecules and Cells

doi: 10.14348/molcells.2020.2283

The N-terminal region of PNPLA7-C, amino acids 681-741, contributes to LD targeting. (A) Schematic diagram of PNPLA7-C mutants tagged with GFP and their colocalization with LD. Mutants failing to localize to LDs were described as negative (–). Those labeled “+” localize to LDs. (B) Subcellular distribution of PNPLA7-C variants. COS-7 were transfected with various PNPLA7-C mutants and LDs were induced by OA loading for 16 h and then fixed. LipidTOX Red was used to stain LDs. Colocalization of various PNPLA7-C-GFP constructs and LDs were visualized by confocal laser scanning microscopy. Scale bars = 10 μm. (C and D) The expression of PNPLA7-C truncation mutants in whole cellular lysates and LD fractions was detected by immunoblotting using GFP antibody. The GAPDH and PLIN2 were used as loading controls.
Figure Legend Snippet: The N-terminal region of PNPLA7-C, amino acids 681-741, contributes to LD targeting. (A) Schematic diagram of PNPLA7-C mutants tagged with GFP and their colocalization with LD. Mutants failing to localize to LDs were described as negative (–). Those labeled “+” localize to LDs. (B) Subcellular distribution of PNPLA7-C variants. COS-7 were transfected with various PNPLA7-C mutants and LDs were induced by OA loading for 16 h and then fixed. LipidTOX Red was used to stain LDs. Colocalization of various PNPLA7-C-GFP constructs and LDs were visualized by confocal laser scanning microscopy. Scale bars = 10 μm. (C and D) The expression of PNPLA7-C truncation mutants in whole cellular lysates and LD fractions was detected by immunoblotting using GFP antibody. The GAPDH and PLIN2 were used as loading controls.

Techniques Used: Labeling, Transfection, Staining, Construct, Confocal Laser Scanning Microscopy, Expressing

The putative four TMDs of PNPLA7-C directs cytosolic GFP to LDs. (A) Schematic diagram of PNPLA7-C truncation mutants and their colocalization with LDs. A “+” indicates the detection of mutated PNPLA7-C on LDs; a “–” indicates the failure to detect the protein on LDs. (B) Subcellular distribution of PNPLA7-C truncation mutants and LDs. COS-7 transiently expressing GFP fused to various PNPLA7-C were incubated with 400 μM OA overnight and then fixed. LD was stained with LipidTOX Red. Colocalization of various PNPLA7-C-GFP constructs and LDs were visualized by confocal fluorescence microscopy. The discontinuous short white lines indicate cell boundaries. Scale bars = 10 μm. (C and D) The expression of PNPLA7-C truncation mutants in whole cell lysis and LD fractions were detected by immunoblotting using GFP antibody. The expression of GAPDH and PLIN2 were used as loading controls.
Figure Legend Snippet: The putative four TMDs of PNPLA7-C directs cytosolic GFP to LDs. (A) Schematic diagram of PNPLA7-C truncation mutants and their colocalization with LDs. A “+” indicates the detection of mutated PNPLA7-C on LDs; a “–” indicates the failure to detect the protein on LDs. (B) Subcellular distribution of PNPLA7-C truncation mutants and LDs. COS-7 transiently expressing GFP fused to various PNPLA7-C were incubated with 400 μM OA overnight and then fixed. LD was stained with LipidTOX Red. Colocalization of various PNPLA7-C-GFP constructs and LDs were visualized by confocal fluorescence microscopy. The discontinuous short white lines indicate cell boundaries. Scale bars = 10 μm. (C and D) The expression of PNPLA7-C truncation mutants in whole cell lysis and LD fractions were detected by immunoblotting using GFP antibody. The expression of GAPDH and PLIN2 were used as loading controls.

Techniques Used: Expressing, Incubation, Staining, Construct, Fluorescence, Microscopy, Lysis

The carboxyl-terminal region of PNPLA7-C blocks LD targeting. (A) Schematic diagram of C-terminal truncation mutants of PNPLA7-C and their colocalization with LDs. Disassociation with LDs is described as negative (–) and localization to LDs as positive (+). (B) Subcellular localization of C-terminal truncated mutants in COS-7 cells. Various PNPLA7-C mutants tagged with GFP were expressed and LDs were induced by 400 μM OA loading for 16 h. LDs was stained with LipidTOX Red. Colocalization of various PNPLA7-C-GFP constructs and the LDs were visualized by confocal fluorescence microscopy. Scale bars = 10 μm. (C and D) The abundance of proteins in whole cellular extracts and LD fractions were assessed by immunoblotting analysis using antibodies against GFP, GAPDH, and PLIN2.
Figure Legend Snippet: The carboxyl-terminal region of PNPLA7-C blocks LD targeting. (A) Schematic diagram of C-terminal truncation mutants of PNPLA7-C and their colocalization with LDs. Disassociation with LDs is described as negative (–) and localization to LDs as positive (+). (B) Subcellular localization of C-terminal truncated mutants in COS-7 cells. Various PNPLA7-C mutants tagged with GFP were expressed and LDs were induced by 400 μM OA loading for 16 h. LDs was stained with LipidTOX Red. Colocalization of various PNPLA7-C-GFP constructs and the LDs were visualized by confocal fluorescence microscopy. Scale bars = 10 μm. (C and D) The abundance of proteins in whole cellular extracts and LD fractions were assessed by immunoblotting analysis using antibodies against GFP, GAPDH, and PLIN2.

Techniques Used: Staining, Construct, Fluorescence, Microscopy

12) Product Images from "DAX1 promotes cervical cancer cell growth and tumorigenicity through activation of Wnt/β-catenin pathway via GSK3β"

Article Title: DAX1 promotes cervical cancer cell growth and tumorigenicity through activation of Wnt/β-catenin pathway via GSK3β

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0359-6

DAX1 expression is shown in normal cervix and different cervical lesions . a Immunohistochemistry showing DAX1 expression in 43 NC, 41 HSIL, and 55 SCC. b The IHC scores of DAX1 staining in NC, HSIL, and SCC were performed. Data were statistically analyzed with the multiple-comparison test of one-way ANOVA and values are shown as mean ± SD. c DAX1 staining is classified into three categories (weak-positive, strong-positive, and negative), and the percentage of each group is shown in total ( c1 ), nucleus ( c2 ), and cytoplasm ( c3 ). d Representative western blots of DAX1 protein expression were detected in NC and SCC. e Quantitative analysis of DAX1 expression in normal cervix and squamous cervical carcinoma; GAPDH was used as an internal control; Student’s t test was carried out. * P
Figure Legend Snippet: DAX1 expression is shown in normal cervix and different cervical lesions . a Immunohistochemistry showing DAX1 expression in 43 NC, 41 HSIL, and 55 SCC. b The IHC scores of DAX1 staining in NC, HSIL, and SCC were performed. Data were statistically analyzed with the multiple-comparison test of one-way ANOVA and values are shown as mean ± SD. c DAX1 staining is classified into three categories (weak-positive, strong-positive, and negative), and the percentage of each group is shown in total ( c1 ), nucleus ( c2 ), and cytoplasm ( c3 ). d Representative western blots of DAX1 protein expression were detected in NC and SCC. e Quantitative analysis of DAX1 expression in normal cervix and squamous cervical carcinoma; GAPDH was used as an internal control; Student’s t test was carried out. * P

Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot

13) Product Images from "A Functional Dissection of PTEN N-Terminus: Implications in PTEN Subcellular Targeting and Tumor Suppressor Activity"

Article Title: A Functional Dissection of PTEN N-Terminus: Implications in PTEN Subcellular Targeting and Tumor Suppressor Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0119287

Functional analysis in mammalian cells of PTEN mutations displaying distinctive nuclear accumulation and PIP3 phosphatase activity. ( A ) The ectopic expression of PTEN wild type (WT) and mutations in U2OS clones was monitored by immunoblot with an anti-PTEN antibody (upper panel). The arrows indicate the migration of the full-length (residues 1–403) or the truncated (residues 1–375) recombinant PTEN. Expression of GAPDH is shown in the lower panel as a loading control. The figure shows non-adjacent bands from the same blot. ( B ) Proliferation of the distinct U2OS clones during 5 days of culture. Data are shown as the mean + SD of the absorbance from three experiments, corrected for background.-, empty vector. ( D ) Growth in soft agar of the distinct U2OS clones. Cells were plated on soft agar, and pictures (X40 magnification) were made after 3–4 weeks of growth (upper part). The bottom graph shows the quantification of the number of colonies per plate for each clone. Data are shown as the mean + SD from three independent experiments. ** p
Figure Legend Snippet: Functional analysis in mammalian cells of PTEN mutations displaying distinctive nuclear accumulation and PIP3 phosphatase activity. ( A ) The ectopic expression of PTEN wild type (WT) and mutations in U2OS clones was monitored by immunoblot with an anti-PTEN antibody (upper panel). The arrows indicate the migration of the full-length (residues 1–403) or the truncated (residues 1–375) recombinant PTEN. Expression of GAPDH is shown in the lower panel as a loading control. The figure shows non-adjacent bands from the same blot. ( B ) Proliferation of the distinct U2OS clones during 5 days of culture. Data are shown as the mean + SD of the absorbance from three experiments, corrected for background.-, empty vector. ( D ) Growth in soft agar of the distinct U2OS clones. Cells were plated on soft agar, and pictures (X40 magnification) were made after 3–4 weeks of growth (upper part). The bottom graph shows the quantification of the number of colonies per plate for each clone. Data are shown as the mean + SD from three independent experiments. ** p

Techniques Used: Functional Assay, Activity Assay, Expressing, Clone Assay, Migration, Recombinant, Plasmid Preparation

14) Product Images from "The C-Terminal Amino Acid of the MHC-I Heavy Chain Is Critical for Binding to Derlin-1 in Human Cytomegalovirus US11-Induced MHC-I Degradation"

Article Title: The C-Terminal Amino Acid of the MHC-I Heavy Chain Is Critical for Binding to Derlin-1 in Human Cytomegalovirus US11-Induced MHC-I Degradation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072356

The C-terminal amino acid of the MHC-I heavy chain is critical for its interaction with Derlin-1 during US11-induced ERAD. (A) Deletion of a single C-terminal amino acid renders HLA-A2 resistant to US11-induced degradation. U373MG-US11 cells were transfected with wild-type HLA-A2, HLA-A2-ΔV or HLA-A2-ΔCT, metabolically labeled for 15 min, and then chased for 0, 45, or 90 min. (B) Deleting the C-terminal amino acid from the GGA construct blocks its degradation by US11. U373MG-US11 cells were transfected with GGA or GGA-ΔV, metabolically labeled for 15 min, and then chased for 0, 45, or 90 min. (C) The C-terminal valine of HLA-A2 is critical for HLA-A2 dislocation to the cytosol during US11-induced degradation. U373MG-US11 cells were transfected with either wild-type HLA-A2 or HLA-A2-ΔV, pulse-labeled for 15 min, and chased for 0 or 60 min. After lysis, HLA-A2 or HLA-A2-ΔV was recovered by immunoprecipitation with mAb BB7.2 and either left untreated or treated with EndoH (upper panel). Comparable levels of GAPDH show that the amount of the cell lysate used was equal between samples (lower panel). MHC-I HC R , EndoH-resistant MHC-I heavy chain; MHC-I HC S , EndoH-sensitive MHC-I heavy chain. (D) Deletion of the C-terminal valine residue disrupts the interaction between GGA and Derlin-1. U373MG-US11 cells were transfected with GGA or GGA-ΔV, metabolically labeled for 1 hr, lysed in 1% digitonin, and subjected to immunoprecipitation with the anti-p97 antibody. The precipitate was then boiled in SDS/DTT-containing buffer, diluted 10-fold in 1% NP-40, and then subjected to a second round of immunoprecipitation with the anti-p97 antibody or with mAb 4H84. (E) Deletion of the C-terminal valine residue of GGA does not affect its interaction with US11. The same digitonin-solubilized lysates described in (D) were subjected to immunoprecipitation with an anti-US11 antibody. The precipitate was then boiled in SDS/DTT-containing buffer, diluted 10-fold in 1% NP-40, and subjected to a second round of immunoprecipitation with the anti-US11 antibody or with mAb 4H84. (F) The C-terminal valine residue of HLA-A2 is not required for HCMV US2-induced degradation. U373MG-US2 cells were transfected with HLA-A2 or HLA-A2-ΔV, metabolically labeled for 15 min, and then chased for 0 or 45 min. HLA-A2 or HLA-A2-ΔV was recovered by immunoprecipitation with mAb BB7.2 and either left untreated or treated with EndoH (upper panel). Similar levels of HSP90 indicate that the amount of the cell lysate used was comparable between samples (lower panel). All experiments were performed multiple times with similar results, and the data shown are representative of all results.
Figure Legend Snippet: The C-terminal amino acid of the MHC-I heavy chain is critical for its interaction with Derlin-1 during US11-induced ERAD. (A) Deletion of a single C-terminal amino acid renders HLA-A2 resistant to US11-induced degradation. U373MG-US11 cells were transfected with wild-type HLA-A2, HLA-A2-ΔV or HLA-A2-ΔCT, metabolically labeled for 15 min, and then chased for 0, 45, or 90 min. (B) Deleting the C-terminal amino acid from the GGA construct blocks its degradation by US11. U373MG-US11 cells were transfected with GGA or GGA-ΔV, metabolically labeled for 15 min, and then chased for 0, 45, or 90 min. (C) The C-terminal valine of HLA-A2 is critical for HLA-A2 dislocation to the cytosol during US11-induced degradation. U373MG-US11 cells were transfected with either wild-type HLA-A2 or HLA-A2-ΔV, pulse-labeled for 15 min, and chased for 0 or 60 min. After lysis, HLA-A2 or HLA-A2-ΔV was recovered by immunoprecipitation with mAb BB7.2 and either left untreated or treated with EndoH (upper panel). Comparable levels of GAPDH show that the amount of the cell lysate used was equal between samples (lower panel). MHC-I HC R , EndoH-resistant MHC-I heavy chain; MHC-I HC S , EndoH-sensitive MHC-I heavy chain. (D) Deletion of the C-terminal valine residue disrupts the interaction between GGA and Derlin-1. U373MG-US11 cells were transfected with GGA or GGA-ΔV, metabolically labeled for 1 hr, lysed in 1% digitonin, and subjected to immunoprecipitation with the anti-p97 antibody. The precipitate was then boiled in SDS/DTT-containing buffer, diluted 10-fold in 1% NP-40, and then subjected to a second round of immunoprecipitation with the anti-p97 antibody or with mAb 4H84. (E) Deletion of the C-terminal valine residue of GGA does not affect its interaction with US11. The same digitonin-solubilized lysates described in (D) were subjected to immunoprecipitation with an anti-US11 antibody. The precipitate was then boiled in SDS/DTT-containing buffer, diluted 10-fold in 1% NP-40, and subjected to a second round of immunoprecipitation with the anti-US11 antibody or with mAb 4H84. (F) The C-terminal valine residue of HLA-A2 is not required for HCMV US2-induced degradation. U373MG-US2 cells were transfected with HLA-A2 or HLA-A2-ΔV, metabolically labeled for 15 min, and then chased for 0 or 45 min. HLA-A2 or HLA-A2-ΔV was recovered by immunoprecipitation with mAb BB7.2 and either left untreated or treated with EndoH (upper panel). Similar levels of HSP90 indicate that the amount of the cell lysate used was comparable between samples (lower panel). All experiments were performed multiple times with similar results, and the data shown are representative of all results.

Techniques Used: Transfection, Metabolic Labelling, Labeling, Construct, Lysis, Immunoprecipitation

15) Product Images from "The homologous recombination protein RAD51 is a promising therapeutic target for cervical carcinoma"

Article Title: The homologous recombination protein RAD51 is a promising therapeutic target for cervical carcinoma

Journal: Oncology Reports

doi: 10.3892/or.2017.5724

RAD51 expression in normal cervix and cervical carcinoma tissues. (A) Immunohistochemistry results showing RAD51 expression in normal cervix and cervical carcinoma tissues; scale bar, 50 µm. (B) RAD51 staining is classified into negative and positive, and the percentage of tissues in each group is shown. (C) The IHC scores of RAD51 staining in the normal cervix and carcinoma tissues are shown. (D) The expression of RAD51 in Caski, C33-A, HeLa and SiHa cells was measured by western blotting, the relative expression of RAD51 was calculated based on western blot analyses, GAPDH was used as an internal control. *P
Figure Legend Snippet: RAD51 expression in normal cervix and cervical carcinoma tissues. (A) Immunohistochemistry results showing RAD51 expression in normal cervix and cervical carcinoma tissues; scale bar, 50 µm. (B) RAD51 staining is classified into negative and positive, and the percentage of tissues in each group is shown. (C) The IHC scores of RAD51 staining in the normal cervix and carcinoma tissues are shown. (D) The expression of RAD51 in Caski, C33-A, HeLa and SiHa cells was measured by western blotting, the relative expression of RAD51 was calculated based on western blot analyses, GAPDH was used as an internal control. *P

Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot

16) Product Images from "Zika virus as an oncolytic treatment of human neuroblastoma cells requires CD24"

Article Title: Zika virus as an oncolytic treatment of human neuroblastoma cells requires CD24

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200358

Analysis of the role of CD24 in Zika-virus infected neuroblastoma cells. A) Western blot analysis of siRNA-mediated knock-down of CD24 expression in IMR-32 cells. Samples include Negative Control siRNA and CD24 siRNA. B) Western blot analysis of the expression of Envelope protein and NS1 (Non-Structural 1) protein in IMR-32 cells after siRNA-mediated knock-down of CD24 expression, 96 hours after Zika infection (MOI = 10). Samples include control cells treated with non-infected conditioned media and infected IMR-32 cells transfected with either Negative Control siRNA or CD24 siRNA. C) Western blot analysis of CD24 expression in the human neuroblastoma cell line SK-N-AS, comparing wild type (WT) to stably selected “Vector Only” (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). D) Western blot analysis of Zika NS1 protein expression 96 hours after Zika infection in CD24-stably expressing SK-N-AS cells, comparing wild type (WT) to stably selected Vector Only (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). GAPDH was used as a load control for all experiments. All results are representative of the combined data of experiments performed in triplicate.
Figure Legend Snippet: Analysis of the role of CD24 in Zika-virus infected neuroblastoma cells. A) Western blot analysis of siRNA-mediated knock-down of CD24 expression in IMR-32 cells. Samples include Negative Control siRNA and CD24 siRNA. B) Western blot analysis of the expression of Envelope protein and NS1 (Non-Structural 1) protein in IMR-32 cells after siRNA-mediated knock-down of CD24 expression, 96 hours after Zika infection (MOI = 10). Samples include control cells treated with non-infected conditioned media and infected IMR-32 cells transfected with either Negative Control siRNA or CD24 siRNA. C) Western blot analysis of CD24 expression in the human neuroblastoma cell line SK-N-AS, comparing wild type (WT) to stably selected “Vector Only” (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). D) Western blot analysis of Zika NS1 protein expression 96 hours after Zika infection in CD24-stably expressing SK-N-AS cells, comparing wild type (WT) to stably selected Vector Only (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). GAPDH was used as a load control for all experiments. All results are representative of the combined data of experiments performed in triplicate.

Techniques Used: Infection, Western Blot, Expressing, Negative Control, Transfection, Stable Transfection, Variant Assay, Plasmid Preparation

Analysis of CD24 expression in human neuroblastoma cells. A) Schematic of the alignment of CD24 splice variants 1 and 7. B C) Absolute quantification of CD24 expression by quantitative real-time PCR of total RNA acquired from neuroblastoma cells, measuring CD24 splice variants 1 (B) and 7 (C). Copy number values were normalized to the corresponding GAPDH values to determine the relative copy number. ** p > 0.05, Student’s t-test. D) Western blot analysis of CD24 expression in the total cell lysates of neuroblastoma cells. GAPDH was used as a loading control. All results are representative of the combined data of experiments performed in triplicate, with error bars representing standard deviation.
Figure Legend Snippet: Analysis of CD24 expression in human neuroblastoma cells. A) Schematic of the alignment of CD24 splice variants 1 and 7. B C) Absolute quantification of CD24 expression by quantitative real-time PCR of total RNA acquired from neuroblastoma cells, measuring CD24 splice variants 1 (B) and 7 (C). Copy number values were normalized to the corresponding GAPDH values to determine the relative copy number. ** p > 0.05, Student’s t-test. D) Western blot analysis of CD24 expression in the total cell lysates of neuroblastoma cells. GAPDH was used as a loading control. All results are representative of the combined data of experiments performed in triplicate, with error bars representing standard deviation.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

17) Product Images from "Centrosomal Protein of 55 Regulates Glucose Metabolism, Proliferation and Apoptosis of Glioma Cells via the Akt/mTOR Signaling Pathway"

Article Title: Centrosomal Protein of 55 Regulates Glucose Metabolism, Proliferation and Apoptosis of Glioma Cells via the Akt/mTOR Signaling Pathway

Journal: Journal of Cancer

doi: 10.7150/jca.15497

CEP55 regulates the Akt/mTOR signaling pathway. (A) Protein levels of p-Akt, total Akt, p-mTOR and total mTOR as determined by Western blot analysis in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55. GAPDH was used as the loading control. (B) Protein levels of Akt downstream targets (BAD, Caspase-9, p27 and GSK-3β) as determined by Western blot analysis in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55.
Figure Legend Snippet: CEP55 regulates the Akt/mTOR signaling pathway. (A) Protein levels of p-Akt, total Akt, p-mTOR and total mTOR as determined by Western blot analysis in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55. GAPDH was used as the loading control. (B) Protein levels of Akt downstream targets (BAD, Caspase-9, p27 and GSK-3β) as determined by Western blot analysis in U87 and T98G cells transfected with control siRNA or siRNA targeting CEP55.

Techniques Used: Western Blot, Transfection

18) Product Images from "Effects of Tiaozhi Granule on Regulation of Autophagy Levels in HUVECs"

Article Title: Effects of Tiaozhi Granule on Regulation of Autophagy Levels in HUVECs

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/1765731

Effects of Tiaozhi granule on rapamycin activated autophagy in HUVECs. (a) Effect of Tiaozhi granule on rapamycin induced Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on rapamycin induced Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on rapamycin induced Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on rapamycin induced mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on rapamycin induced LC3 protein cleavage (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on rapamycin induced Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on rapamycin induced p62 protein degradation (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗ P
Figure Legend Snippet: Effects of Tiaozhi granule on rapamycin activated autophagy in HUVECs. (a) Effect of Tiaozhi granule on rapamycin induced Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on rapamycin induced Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on rapamycin induced Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on rapamycin induced mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on rapamycin induced LC3 protein cleavage (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on rapamycin induced Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on rapamycin induced p62 protein degradation (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗ P

Techniques Used: Expressing

Effects of Tiaozhi granule on autophagy level in HUVECs. (a) Effect of Tiaozhi granule on Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on LC3 protein expression (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on p62 protein expression (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗ P
Figure Legend Snippet: Effects of Tiaozhi granule on autophagy level in HUVECs. (a) Effect of Tiaozhi granule on Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on LC3 protein expression (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on p62 protein expression (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗ P

Techniques Used: Expressing

Effects of Tiaozhi granule on Ang II-induced autophagy in HUVECs. (a) Effect of Tiaozhi granule on Ang II-induced Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on Ang II-induced Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on Ang II-induced Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on Ang II-induced mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on Ang II-induced LC3 protein cleavage (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on Ang II-induced Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on Ang II-induced p62 protein degradation (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗ P
Figure Legend Snippet: Effects of Tiaozhi granule on Ang II-induced autophagy in HUVECs. (a) Effect of Tiaozhi granule on Ang II-induced Atg5 mRNA expression (n = 6). (b) Effect of Tiaozhi granule on Ang II-induced Atg7 mRNA expression (n = 6). (c) Effect of Tiaozhi granule on Ang II-induced Beclin-1 mRNA expression (n = 6). (d) Effect of Tiaozhi granule on Ang II-induced mTOR mRNA expression (n = 6). (e) Effect of Tiaozhi granule on Ang II-induced LC3 protein cleavage (n = 6). Upper part is representative blot of LC3 and GAPDH; lower part is the densitometric analysis of the ratio of LC3-II to LC3-I normalized to GAPDH. (f) Effect of Tiaozhi granule on Ang II-induced Beclin-1 protein expression (n = 6). Upper part is representative blot of Beclin-1 and GAPDH; lower part is the densitometric analysis of Beclin-1 expression normalized to GAPDH. (g) Effect of Tiaozhi granule on Ang II-induced p62 protein degradation (n = 6). Upper part is representative blot of p62 and GAPDH; lower part is the densitometric analysis of the ratio of p62 expression normalized to GAPDH. Data were presented as mean ± SEM. ∗ P

Techniques Used: Expressing

19) Product Images from "p53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1 and ZEB2"

Article Title: p53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1 and ZEB2

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20110235

miR-200 family members are directly regulated by p53 at the transcriptional level. (A) sqRT-PCR of miR-200 family clusters. The regions (A–F) amplified by sqRT-PCR are indicated with blue-colored bars in a schematic (top). (B) Schematic view of miRNA-200 family clusters and chromosomal locations of TSS and putative p53BS (blue ovals: 200c1, 200c2, and 200b). Gray and white fragments represent 500 bp in length. Small gray arrows indicate primers (F, forward; R, reverse) used for the constructs of luciferase assay (D). Large black arrows indicate locations of precursor miRNAs. (C) ChIP analysis using C3A cells. 200c1 and 200c2 designate p53BSs of miR-200c–141 cluster and 200b designates p53BS of miR-200b–429 cluster. 200c AR designates a region adjacent (∼200 bp from 200c2) to p53BS of miR-200c–141 cluster. 200c AR and GAPDH are used as negative controls. Input represents 1% of the total chromatin used in this analysis. (D) Luciferase assay of 5′ promoters of miR-200 family clusters in C3A-sh-CTRL and C3A-sh-p53 cells. The regions inserted into luciferase reporters are depicted in B. RL, Renilla luciferase; FL, Firefly luciferase. (E, top) Luciferase assay of 5′ promoter with WT or mutant (MUT) p53BSs of miR-200 family clusters in C3A-sh-CTRL and C3A-sh-p53 cells. (bottom) Sequence information on p53BSs mutations. (A and C) A representative experiment out of two independent experiments. (D and E) Data are mean ± SEM of three independent experiments and each is measured in triplicate (**, P
Figure Legend Snippet: miR-200 family members are directly regulated by p53 at the transcriptional level. (A) sqRT-PCR of miR-200 family clusters. The regions (A–F) amplified by sqRT-PCR are indicated with blue-colored bars in a schematic (top). (B) Schematic view of miRNA-200 family clusters and chromosomal locations of TSS and putative p53BS (blue ovals: 200c1, 200c2, and 200b). Gray and white fragments represent 500 bp in length. Small gray arrows indicate primers (F, forward; R, reverse) used for the constructs of luciferase assay (D). Large black arrows indicate locations of precursor miRNAs. (C) ChIP analysis using C3A cells. 200c1 and 200c2 designate p53BSs of miR-200c–141 cluster and 200b designates p53BS of miR-200b–429 cluster. 200c AR designates a region adjacent (∼200 bp from 200c2) to p53BS of miR-200c–141 cluster. 200c AR and GAPDH are used as negative controls. Input represents 1% of the total chromatin used in this analysis. (D) Luciferase assay of 5′ promoters of miR-200 family clusters in C3A-sh-CTRL and C3A-sh-p53 cells. The regions inserted into luciferase reporters are depicted in B. RL, Renilla luciferase; FL, Firefly luciferase. (E, top) Luciferase assay of 5′ promoter with WT or mutant (MUT) p53BSs of miR-200 family clusters in C3A-sh-CTRL and C3A-sh-p53 cells. (bottom) Sequence information on p53BSs mutations. (A and C) A representative experiment out of two independent experiments. (D and E) Data are mean ± SEM of three independent experiments and each is measured in triplicate (**, P

Techniques Used: Polymerase Chain Reaction, Amplification, Construct, Luciferase, Chromatin Immunoprecipitation, Mutagenesis, Sequencing

20) Product Images from "Midkine-Deficiency Delays Chondrogenesis during the Early Phase of Fracture Healing in Mice"

Article Title: Midkine-Deficiency Delays Chondrogenesis during the Early Phase of Fracture Healing in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0116282

Mdk is expressed during ATDC5 cell differentiation and Mdk knockdown significantly delayed early chondrogenic differentiation via suppression of Wnt-target genes. ATDC5 cells were differentiated and gene expression was evaluated using real-time RT-PCR. B2M was used as the housekeeping gene and gene expression values were normalized to the pre-differentiation values (dotted line). ATDC5 cells were incubated in differentiation medium for 5, 7 and 10 days and A) Mdk and B) collagen2a1 gene expression was evaluated using real time RT-PCR. C) ATDC5 cells were incubated with control siRNA or Mdk siRNA for 24 h and subsequently differentiated for 5 days. Mdk knockdown was verified by analyzing Mdk gene expression. Differentiation was analyzed by evaluation of D) aggrecan or E) collagen2a1 gene expression. Beta-catenin signaling was analyzed by evaluation of F) lef1 and G) axin2 gene expression. H) Western blot analysis of Mdk, collagen type 2 and beta-catenin protein expression at days 0 and 5 of differentiation. GAPDH was used as control. *Significantly different from the control values (p
Figure Legend Snippet: Mdk is expressed during ATDC5 cell differentiation and Mdk knockdown significantly delayed early chondrogenic differentiation via suppression of Wnt-target genes. ATDC5 cells were differentiated and gene expression was evaluated using real-time RT-PCR. B2M was used as the housekeeping gene and gene expression values were normalized to the pre-differentiation values (dotted line). ATDC5 cells were incubated in differentiation medium for 5, 7 and 10 days and A) Mdk and B) collagen2a1 gene expression was evaluated using real time RT-PCR. C) ATDC5 cells were incubated with control siRNA or Mdk siRNA for 24 h and subsequently differentiated for 5 days. Mdk knockdown was verified by analyzing Mdk gene expression. Differentiation was analyzed by evaluation of D) aggrecan or E) collagen2a1 gene expression. Beta-catenin signaling was analyzed by evaluation of F) lef1 and G) axin2 gene expression. H) Western blot analysis of Mdk, collagen type 2 and beta-catenin protein expression at days 0 and 5 of differentiation. GAPDH was used as control. *Significantly different from the control values (p

Techniques Used: Cell Differentiation, Expressing, Quantitative RT-PCR, Incubation, Western Blot

21) Product Images from "Discoidin domain receptor 1 controls linear invadosome formation via a Cdc42–Tuba pathway"

Article Title: Discoidin domain receptor 1 controls linear invadosome formation via a Cdc42–Tuba pathway

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201404079

DDR1 localizes at linear invadosomes and is required for their formation. (A, top) MDA-MB-231 cells transiently transfected with DDR1-Flag were cultured for 4 h on collagen I. All channels of the boxed region are shown magnified on the right. F-actin (red) colocalizes with DDR1 (green) and Tks5 (blue) at linear invadosomes. (A, bottom) MDA-MB-231 cells stably expressing DDR1-GFP were cultured for 4 h on collagen I fibrils. Tks5 (red) colocalizes with DDR1 (green) and collagen I (blue). Correlation coefficient of colocalization: actin/DDR1 r = 0.29; DDR1/Tks5 r = 0.11; n = 10. (B) MDA-MB-231 cells were transfected with control (siCT) or three independent DDR1 siRNAs. DDR1 protein expression was analyzed by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Cells transfected as in B were seeded for 4 h on collagen I. Shown are representative confocal images of MDA-MB-231 cells. Tks5 (green) and F-actin (red) are shown. Right panels show enlarged views of the boxed regions. Similar results were obtained with siDDR1 #2 and #3. (D–F) Down-regulation of DDR1 expression decreases the formation of linear invadosomes and their degradation activity. (D) Quantification of the percentage of MDA-MB-231 cells able to form linear invadosomes. Error bars represent the SEM ( n > 1,000; three independent experiments; ***, P
Figure Legend Snippet: DDR1 localizes at linear invadosomes and is required for their formation. (A, top) MDA-MB-231 cells transiently transfected with DDR1-Flag were cultured for 4 h on collagen I. All channels of the boxed region are shown magnified on the right. F-actin (red) colocalizes with DDR1 (green) and Tks5 (blue) at linear invadosomes. (A, bottom) MDA-MB-231 cells stably expressing DDR1-GFP were cultured for 4 h on collagen I fibrils. Tks5 (red) colocalizes with DDR1 (green) and collagen I (blue). Correlation coefficient of colocalization: actin/DDR1 r = 0.29; DDR1/Tks5 r = 0.11; n = 10. (B) MDA-MB-231 cells were transfected with control (siCT) or three independent DDR1 siRNAs. DDR1 protein expression was analyzed by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Cells transfected as in B were seeded for 4 h on collagen I. Shown are representative confocal images of MDA-MB-231 cells. Tks5 (green) and F-actin (red) are shown. Right panels show enlarged views of the boxed regions. Similar results were obtained with siDDR1 #2 and #3. (D–F) Down-regulation of DDR1 expression decreases the formation of linear invadosomes and their degradation activity. (D) Quantification of the percentage of MDA-MB-231 cells able to form linear invadosomes. Error bars represent the SEM ( n > 1,000; three independent experiments; ***, P

Techniques Used: Multiple Displacement Amplification, Transfection, Cell Culture, Stable Transfection, Expressing, Activity Assay

Cdc42 drives linear invadosome formation via DDR1. (A) Western blot analysis of MDA-MB-231 cells transfected with siRNA control (siCT) or two independent siRNAs targeting Rac1, Cdc42, or RhoA. GAPDH is used as a loading control. Three independent experiments were realized and quantified to demonstrate a specific effect on targeted RhoGTPase expression represented on the bar graph on the right. (B and C) MDA-MB-231 cells transfected as in A were cultured on collagen I for 4 h, fixed, and processed for immunofluorescence staining. (B) Representative confocal images of MDA-MB-231 cells transfected as in A. Tks5 (green) and F-actin (red) are shown. Panels on the right show enlarged views of the boxed regions. Bars: (left) 10 µm; (enlarged panels on the right) 2.5 µm. (C) The percentage of siRNA-transfected MDA-MB-231 cells able to form linear invadosomes was quantified. Error bars represent the SEM ( n > 1,000, three independent experiments; ***, P
Figure Legend Snippet: Cdc42 drives linear invadosome formation via DDR1. (A) Western blot analysis of MDA-MB-231 cells transfected with siRNA control (siCT) or two independent siRNAs targeting Rac1, Cdc42, or RhoA. GAPDH is used as a loading control. Three independent experiments were realized and quantified to demonstrate a specific effect on targeted RhoGTPase expression represented on the bar graph on the right. (B and C) MDA-MB-231 cells transfected as in A were cultured on collagen I for 4 h, fixed, and processed for immunofluorescence staining. (B) Representative confocal images of MDA-MB-231 cells transfected as in A. Tks5 (green) and F-actin (red) are shown. Panels on the right show enlarged views of the boxed regions. Bars: (left) 10 µm; (enlarged panels on the right) 2.5 µm. (C) The percentage of siRNA-transfected MDA-MB-231 cells able to form linear invadosomes was quantified. Error bars represent the SEM ( n > 1,000, three independent experiments; ***, P

Techniques Used: Western Blot, Multiple Displacement Amplification, Transfection, Expressing, Cell Culture, Immunofluorescence, Staining

Linear invadosome formation and activity is independent of Src activity. (A and B) MDA-MB-231 cells were seeded on gelatin-FITC (top) or on a mixed matrix (collagen I/gelatin-FITC; bottom) and treated with 5 µM PP2 (Src inhibitor) or DMSO (vehicle). Gelatin (gray), Tks5 (green), and F-actin (red) are shown. (C) Quantification of the degradation capacity of MDA-MB-231 cells seeded on a mixed gelatin/collagen I matrix treated or not treated with PP2. The left graph represents the gelatin area degraded per cell after 24 h. The right graph represents the amount of collagen degraded after 4 h ( n = 30 fields). Data are shown as mean ± SEM of three independent experiments. (D) MDA-MB-231 cells transfected with control (siCT) or two independent DDR1 siRNAs (DDR1 #1 and #2) were seeded on gelatin or collagen I and treated with PP2. Protein extracts were then analyzed by immunoblotting to determine phospho-Src, total Src, and DDR1 protein expression (representative of three experiments). (E and F) Western blots performed on SYF and SYF c-Src protein extracts representing, respectively, protein expression of Src and DDR1. GAPDH was used as a loading control. (G and H) Confocal images of control cells (SYF c-Src) and SYF fibroblasts cultured on gelatin (G) or on a mixed matrix (gelatin/collagen I; H) for 24 h and processed for immunofluorescence staining (F-actin, red; Tks5, green; DAPI, blue). Insets on the bottom show gelatin-degraded pictures. Bars: (A, G, and H) 10 µm; (A, insets) 7 µm; (H, top insets) 2 µm; (G and H, bottom insets) 10 µm.
Figure Legend Snippet: Linear invadosome formation and activity is independent of Src activity. (A and B) MDA-MB-231 cells were seeded on gelatin-FITC (top) or on a mixed matrix (collagen I/gelatin-FITC; bottom) and treated with 5 µM PP2 (Src inhibitor) or DMSO (vehicle). Gelatin (gray), Tks5 (green), and F-actin (red) are shown. (C) Quantification of the degradation capacity of MDA-MB-231 cells seeded on a mixed gelatin/collagen I matrix treated or not treated with PP2. The left graph represents the gelatin area degraded per cell after 24 h. The right graph represents the amount of collagen degraded after 4 h ( n = 30 fields). Data are shown as mean ± SEM of three independent experiments. (D) MDA-MB-231 cells transfected with control (siCT) or two independent DDR1 siRNAs (DDR1 #1 and #2) were seeded on gelatin or collagen I and treated with PP2. Protein extracts were then analyzed by immunoblotting to determine phospho-Src, total Src, and DDR1 protein expression (representative of three experiments). (E and F) Western blots performed on SYF and SYF c-Src protein extracts representing, respectively, protein expression of Src and DDR1. GAPDH was used as a loading control. (G and H) Confocal images of control cells (SYF c-Src) and SYF fibroblasts cultured on gelatin (G) or on a mixed matrix (gelatin/collagen I; H) for 24 h and processed for immunofluorescence staining (F-actin, red; Tks5, green; DAPI, blue). Insets on the bottom show gelatin-degraded pictures. Bars: (A, G, and H) 10 µm; (A, insets) 7 µm; (H, top insets) 2 µm; (G and H, bottom insets) 10 µm.

Techniques Used: Activity Assay, Multiple Displacement Amplification, Transfection, Expressing, Western Blot, Cell Culture, Immunofluorescence, Staining

22) Product Images from "The nuclear cofactor RAC3/AIB1/SRC-3 enhances Nrf2 signaling by interacting with transactivation domains"

Article Title: The nuclear cofactor RAC3/AIB1/SRC-3 enhances Nrf2 signaling by interacting with transactivation domains

Journal: Oncogene

doi: 10.1038/onc.2012.59

Nrf2 binds directly to the RAC3 protein A) Prior to the co-IP study with Nrf2 and RAC3, the two constructs were verified by determining EGFP-Nrf2 and HA-RAC3 expression levels in HeLa cells. The cells were transiently transfected with GFP-Nrf2 (3 μg) or HA-RAC3 (3 μg) using jetPEI reagent (Polyplus-Transfection) for 24 h followed by treatment with MG132, a proteasome inhibitor, for 6 h. The protein samples (20 μg) were subjected to western blot analysis for Nrf2 and RAC3 using anti-GFP or anti-Nrf2 (C-20) and anti-HA or anti-RAC3 (M-397) antibodies. Actin expression was detected to confirm equal loading. B) To determine the subcellular localization of RAC3 and Nrf2 in HeLa cells, MG132 (10 μM) was administered for different times, and the cytosolic and nuclear fractions were isolated using M-PER buffer (Pierce). The fractionated samples (20 μg) were subjected to western blot analysis to measure endogenous protein levels using specific antibodies as indicated. Lamin A was used as the positive control for the nuclear fraction. C) The cellular co-localization of endogenous Nrf2 and RAC3 in HeLa cells was visualized using immunofluorescence microscopy after DL-sulforaphane (SFN, 20 μM) treatment for 16 h. Anti-Nrf2 (Epitomics, California, Burlingame, USA) and an Alexa Fluor 594 secondary antibody was used to visualize Nrf2. Anti-RAC3 (E-11) and an Alexa Fluor 488 secondary antibody was used to visualize RAC3. Magnification, 100X. D) To confirm the binding interaction between Nrf2 and RAC3, whole-cell lysates from MCF7 cells were subjected to the IP of endogenous Nrf2 using an anti-Nrf2 antibody followed by western blotting against endogenous RAC3 using an anti-RAC3 antibody. E) To determine whether Nrf2 could bind to the RAC3 protein, HeLa cells in 6-well plates were transfected with EGFP-Nrf2 (2 μg) and HA-RAC3 (2 μg) constructs for 24 h, and a co-IP assay was performed. A total of 200 μg of protein from the different fractions were immunoprecipitated using anti-RAC3 (M-397) and blotted for EGFP-Nrf2 using an anti-GFP antibody using western blot analysis. The co-IP method is described in the Materials and Methods. GAPDH and Lamin A were used as the controls for the cytosolic and nuclear fractions, respectively. The IgG heavy chain was used to confirm equal bead loading. F) To determine whether Nrf2 could directly bind to RAC3, purified His-Nrf2 and GST-RAC3 expressed in a bacterial expression system were co-incubated, and GST-RAC3 was pulled down using GSH beads in vitro. The protein-bead complexes were subjected to western blot analysis using an anti-Nrf2 (C-20) antibody. The detailed procedures are described in the Materials and Methods. Asterisks indicate the predicted size of the GST-RAC3 protein.
Figure Legend Snippet: Nrf2 binds directly to the RAC3 protein A) Prior to the co-IP study with Nrf2 and RAC3, the two constructs were verified by determining EGFP-Nrf2 and HA-RAC3 expression levels in HeLa cells. The cells were transiently transfected with GFP-Nrf2 (3 μg) or HA-RAC3 (3 μg) using jetPEI reagent (Polyplus-Transfection) for 24 h followed by treatment with MG132, a proteasome inhibitor, for 6 h. The protein samples (20 μg) were subjected to western blot analysis for Nrf2 and RAC3 using anti-GFP or anti-Nrf2 (C-20) and anti-HA or anti-RAC3 (M-397) antibodies. Actin expression was detected to confirm equal loading. B) To determine the subcellular localization of RAC3 and Nrf2 in HeLa cells, MG132 (10 μM) was administered for different times, and the cytosolic and nuclear fractions were isolated using M-PER buffer (Pierce). The fractionated samples (20 μg) were subjected to western blot analysis to measure endogenous protein levels using specific antibodies as indicated. Lamin A was used as the positive control for the nuclear fraction. C) The cellular co-localization of endogenous Nrf2 and RAC3 in HeLa cells was visualized using immunofluorescence microscopy after DL-sulforaphane (SFN, 20 μM) treatment for 16 h. Anti-Nrf2 (Epitomics, California, Burlingame, USA) and an Alexa Fluor 594 secondary antibody was used to visualize Nrf2. Anti-RAC3 (E-11) and an Alexa Fluor 488 secondary antibody was used to visualize RAC3. Magnification, 100X. D) To confirm the binding interaction between Nrf2 and RAC3, whole-cell lysates from MCF7 cells were subjected to the IP of endogenous Nrf2 using an anti-Nrf2 antibody followed by western blotting against endogenous RAC3 using an anti-RAC3 antibody. E) To determine whether Nrf2 could bind to the RAC3 protein, HeLa cells in 6-well plates were transfected with EGFP-Nrf2 (2 μg) and HA-RAC3 (2 μg) constructs for 24 h, and a co-IP assay was performed. A total of 200 μg of protein from the different fractions were immunoprecipitated using anti-RAC3 (M-397) and blotted for EGFP-Nrf2 using an anti-GFP antibody using western blot analysis. The co-IP method is described in the Materials and Methods. GAPDH and Lamin A were used as the controls for the cytosolic and nuclear fractions, respectively. The IgG heavy chain was used to confirm equal bead loading. F) To determine whether Nrf2 could directly bind to RAC3, purified His-Nrf2 and GST-RAC3 expressed in a bacterial expression system were co-incubated, and GST-RAC3 was pulled down using GSH beads in vitro. The protein-bead complexes were subjected to western blot analysis using an anti-Nrf2 (C-20) antibody. The detailed procedures are described in the Materials and Methods. Asterisks indicate the predicted size of the GST-RAC3 protein.

Techniques Used: Co-Immunoprecipitation Assay, Construct, Expressing, Transfection, Western Blot, Isolation, Positive Control, Immunofluorescence, Microscopy, Binding Assay, Immunoprecipitation, Purification, Incubation, In Vitro

23) Product Images from "Octacosanol and policosanol prevent high-fat diet-induced obesity and metabolic disorders by activating brown adipose tissue and improving liver metabolism"

Article Title: Octacosanol and policosanol prevent high-fat diet-induced obesity and metabolic disorders by activating brown adipose tissue and improving liver metabolism

Journal: Scientific Reports

doi: 10.1038/s41598-019-41631-1

Effect of octacosanol and policosanol on inguinal white adipose tissue (iWAT) of mice fed on chow, HFD and HFD treated with octacosanol or policosanol. ( a ) Representative H E-stained sections of iWAT harvested from mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks. Scale bar = 30 μm. ( b ) Average adipocyte size in iWAT of mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks (n = 5). ( c ) qRT-PCR analysis of beige fat markers in iWAT harvested from mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks (n = 5–8). ( d ) Western blotting of UCP-1 and GAPDH in iWAT (n = 4–5). GAPDH was used as a loading control. ( e ) Protein expression of UCP-1 determined by densitometry analysis (n = 4–5). Values represent mean ± SEM * P
Figure Legend Snippet: Effect of octacosanol and policosanol on inguinal white adipose tissue (iWAT) of mice fed on chow, HFD and HFD treated with octacosanol or policosanol. ( a ) Representative H E-stained sections of iWAT harvested from mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks. Scale bar = 30 μm. ( b ) Average adipocyte size in iWAT of mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks (n = 5). ( c ) qRT-PCR analysis of beige fat markers in iWAT harvested from mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks (n = 5–8). ( d ) Western blotting of UCP-1 and GAPDH in iWAT (n = 4–5). GAPDH was used as a loading control. ( e ) Protein expression of UCP-1 determined by densitometry analysis (n = 4–5). Values represent mean ± SEM * P

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

Effect of octacosanol and policosanol on brown adipose tissue (BAT) of mice fed on chow, HFD and HFD treated with octacosanol or policosanol. ( a ) Representative hematoxylin and eosin (H E)-stained sections of BAT harvested from mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks. ( b ) Western blotting of UCP-1 and GAPDH in BAT (n = 5). GAPDH was used as a loading control. ( c ) Protein expression of UCP-1 determined by densitometry analysis (n = 5). ( d ) Quantitative real-time PCR (qRT-PCR) analysis of genes involved in thermogenesis of BAT in mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks (n = 5–8). ( e ) Rectal temperature of mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks (n = 5). Values represent mean ± SEM. * P
Figure Legend Snippet: Effect of octacosanol and policosanol on brown adipose tissue (BAT) of mice fed on chow, HFD and HFD treated with octacosanol or policosanol. ( a ) Representative hematoxylin and eosin (H E)-stained sections of BAT harvested from mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks. ( b ) Western blotting of UCP-1 and GAPDH in BAT (n = 5). GAPDH was used as a loading control. ( c ) Protein expression of UCP-1 determined by densitometry analysis (n = 5). ( d ) Quantitative real-time PCR (qRT-PCR) analysis of genes involved in thermogenesis of BAT in mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks (n = 5–8). ( e ) Rectal temperature of mice fed on chow, HFD and HFD treated with octacosanol or policosanol for four weeks (n = 5). Values represent mean ± SEM. * P

Techniques Used: Mouse Assay, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

24) Product Images from "A De NovoRAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution"

Article Title: A De NovoRAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution

Journal: Experimental Neurobiology

doi: 10.5607/en.2018.27.6.550

ALS patient-derived fibroblasts display reduced levels of acetylated α-tubulin and disrupted microtubule network. (A) Confocal images of control-derived fibroblasts and ALS patient-derived fibroblasts (E1357K) labeled with antibodies against acetylated α-tubulin (Ac-tub), tyrosinated α-tubulin (Tyr-tub), or α-tubulin (Tub) and DAPI. Scale bar, 5 µm. (B) Western blot analysis of lysates from control and patient fibroblasts using anti-acetylated α-tubulin, anti-tyrosinated α-tubulin, anti-α-tubulin, and anti-GAPDH antibodies. (C and D) Quantitative analysis by densitometric measurements (n=3). The intensities of Ac-tub and Tyr-tub were normalized to that of Tub. Data are presented as mean±SEM. * p
Figure Legend Snippet: ALS patient-derived fibroblasts display reduced levels of acetylated α-tubulin and disrupted microtubule network. (A) Confocal images of control-derived fibroblasts and ALS patient-derived fibroblasts (E1357K) labeled with antibodies against acetylated α-tubulin (Ac-tub), tyrosinated α-tubulin (Tyr-tub), or α-tubulin (Tub) and DAPI. Scale bar, 5 µm. (B) Western blot analysis of lysates from control and patient fibroblasts using anti-acetylated α-tubulin, anti-tyrosinated α-tubulin, anti-α-tubulin, and anti-GAPDH antibodies. (C and D) Quantitative analysis by densitometric measurements (n=3). The intensities of Ac-tub and Tyr-tub were normalized to that of Tub. Data are presented as mean±SEM. * p

Techniques Used: Derivative Assay, Labeling, Western Blot

25) Product Images from "KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex"

Article Title: KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193644

Molecular analysis of in vivo tumor samples. A. Electropherogram analyze % of mu and wt KRAS transcripts in in vivo treated tumor samples. Tumors were removed from animals after four weeks of various treatments, proportions of mu and wt KRAS transcripts were analyzed by RFLP and assayed by Experion. Sample designation is the same as indicated for Fig 4B . % mu and wt KRAS transcripts show at the bottom of the figure was determined by Experion software. B. Western transfer shows protein expression in various tumor samples. Numbers on each sample indicate treatment groups as presented in Fig 4 . Two independent isolated tumors are analyzed for each treatment group. Panel a shows RAS protein in various treated tumor samples normalized against GAPDH. Panel b shows p-EGFR at position Y1068 quantitatively normalized against total EGFR protein. Panel c shows total EGFR protein for various treatment groups. C. Bar graphs summarize fold intensity difference from various groups of in vivo samples. Sample groupings are the same as shown on Fig 4 . Panel a is for p-EGFR at Y1045 normalized to total EGFR protein. Panel b is for p-EGFR at Y1068 normalized to total EGFR protein. Panel c is for p-EGFR at Y1125 normalized to total EGFR protein. Panel d is total EGFR protein normalized to GAPDH. For each sample n = 3. Bar graphs shown are data obtained from approximately half of tumor of three independent animals. Standard deviation bar represents measurements of tumor from three animals. With one tailed, equal variances, student T-test, the following samples show statistical significant ρ-value ≤ 0.05: Panel a between samples 1 and 6, Panel c between samples 1 and 5, Panel d between samples 1 and 4, 2 and 4, 2 and 6.
Figure Legend Snippet: Molecular analysis of in vivo tumor samples. A. Electropherogram analyze % of mu and wt KRAS transcripts in in vivo treated tumor samples. Tumors were removed from animals after four weeks of various treatments, proportions of mu and wt KRAS transcripts were analyzed by RFLP and assayed by Experion. Sample designation is the same as indicated for Fig 4B . % mu and wt KRAS transcripts show at the bottom of the figure was determined by Experion software. B. Western transfer shows protein expression in various tumor samples. Numbers on each sample indicate treatment groups as presented in Fig 4 . Two independent isolated tumors are analyzed for each treatment group. Panel a shows RAS protein in various treated tumor samples normalized against GAPDH. Panel b shows p-EGFR at position Y1068 quantitatively normalized against total EGFR protein. Panel c shows total EGFR protein for various treatment groups. C. Bar graphs summarize fold intensity difference from various groups of in vivo samples. Sample groupings are the same as shown on Fig 4 . Panel a is for p-EGFR at Y1045 normalized to total EGFR protein. Panel b is for p-EGFR at Y1068 normalized to total EGFR protein. Panel c is for p-EGFR at Y1125 normalized to total EGFR protein. Panel d is total EGFR protein normalized to GAPDH. For each sample n = 3. Bar graphs shown are data obtained from approximately half of tumor of three independent animals. Standard deviation bar represents measurements of tumor from three animals. With one tailed, equal variances, student T-test, the following samples show statistical significant ρ-value ≤ 0.05: Panel a between samples 1 and 6, Panel c between samples 1 and 5, Panel d between samples 1 and 4, 2 and 4, 2 and 6.

Techniques Used: In Vivo, Software, Western Blot, Expressing, Isolation, Standard Deviation, One-tailed Test

26) Product Images from "Leucine‐rich α‐2 glycoprotein promotes lung fibrosis by modulating TGF‐β signaling in fibroblasts. Leucine‐rich α‐2 glycoprotein promotes lung fibrosis by modulating TGF‐β signaling in fibroblasts"

Article Title: Leucine‐rich α‐2 glycoprotein promotes lung fibrosis by modulating TGF‐β signaling in fibroblasts. Leucine‐rich α‐2 glycoprotein promotes lung fibrosis by modulating TGF‐β signaling in fibroblasts

Journal: Physiological Reports

doi: 10.14814/phy2.13556

Effect of LRG on TGF ‐ β ‐induced Smad1/5/8 phosphorylation and downstream gene expression in L929. (A) qPCR analysis of Id1 . L929 cells were treated with 2 ng/ mL of TGF ‐ β 1 for 1, 3, and 6 h. The relative expression of Id1 , normalized to Hprt1 gene expression is shown. (B) Detection of phospho‐Smad1/5/8 and Id1 by western blot analysis. L929 cells were transfected with endoglin si RNA and control si RNA and then treated with 2 ng/ mL of TGF ‐ β 1 for 0, 30, and 60 min. Endoglin, phospho‐Smad1/5 (Ser463/465)/8 (Ser426/428), Id1, LRG , and GAPDH were detected by western blot analysis.
Figure Legend Snippet: Effect of LRG on TGF ‐ β ‐induced Smad1/5/8 phosphorylation and downstream gene expression in L929. (A) qPCR analysis of Id1 . L929 cells were treated with 2 ng/ mL of TGF ‐ β 1 for 1, 3, and 6 h. The relative expression of Id1 , normalized to Hprt1 gene expression is shown. (B) Detection of phospho‐Smad1/5/8 and Id1 by western blot analysis. L929 cells were transfected with endoglin si RNA and control si RNA and then treated with 2 ng/ mL of TGF ‐ β 1 for 0, 30, and 60 min. Endoglin, phospho‐Smad1/5 (Ser463/465)/8 (Ser426/428), Id1, LRG , and GAPDH were detected by western blot analysis.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection

Contribution of endoglin to the effect of LRG on TGF ‐ β ‐Smad2 signaling in fibroblasts. (A) (Left) Detection of endoglin in L929 cells by western blot analysis. Cells were transfected with endoglin si RNA or control si RNA and incubated for 24 h. GAPDH was used as an internal control. (Middle and right) qPCR analysis of Serpine1 and Id1 . L929 cells were treated with 2 ng/mL of TGF‐ β 1 for 6 h for Serpine1 or for 1 h for Id1 . The relative expressions of Serpine1 and Id1 , normalized to Hprt1 gene expression are shown. (B) qPCR analysis of Serpine1 and Acta2 . L929 cells were transfected with endoglin siRNA or control siRNA and then treated with 2 ng/mL of TGF‐ β 1 for 6 h for Serpine1 or 12 h for Acta2 . The relative expressions of Serpine1 and Acta2 , normalized to Hprt1 gene expression are shown. Two‐way ANOVA followed by Tukey's test was used for statistical analysis. (C) Detection of endoglin in NIH/3T3 cells by western blot analysis. Cells were treated with 2 ng/mL of TGF‐ β and/or 20 μ g/mL of LRG for 24 h. L929 treated with 2 ng/mL of TGF‐ β for 60 min was used as a positive control for endoglin and pSmad2. (D and E) Detection of phospho‐Smad2 in parental NIH/3T3 cells. Cells were treated with 20 μ g/mL of affinity‐purified LRG for 24 h and then treated with 2 ng/mL of TGF‐ β 1 for 30 min. Phospho‐Smad2, Smad2, LRG, and GAPDH were detected by western blot analysis. Band intensity of pSmad2 normalized to Smad2 is shown in Figure 5 E ( n = 3).
Figure Legend Snippet: Contribution of endoglin to the effect of LRG on TGF ‐ β ‐Smad2 signaling in fibroblasts. (A) (Left) Detection of endoglin in L929 cells by western blot analysis. Cells were transfected with endoglin si RNA or control si RNA and incubated for 24 h. GAPDH was used as an internal control. (Middle and right) qPCR analysis of Serpine1 and Id1 . L929 cells were treated with 2 ng/mL of TGF‐ β 1 for 6 h for Serpine1 or for 1 h for Id1 . The relative expressions of Serpine1 and Id1 , normalized to Hprt1 gene expression are shown. (B) qPCR analysis of Serpine1 and Acta2 . L929 cells were transfected with endoglin siRNA or control siRNA and then treated with 2 ng/mL of TGF‐ β 1 for 6 h for Serpine1 or 12 h for Acta2 . The relative expressions of Serpine1 and Acta2 , normalized to Hprt1 gene expression are shown. Two‐way ANOVA followed by Tukey's test was used for statistical analysis. (C) Detection of endoglin in NIH/3T3 cells by western blot analysis. Cells were treated with 2 ng/mL of TGF‐ β and/or 20 μ g/mL of LRG for 24 h. L929 treated with 2 ng/mL of TGF‐ β for 60 min was used as a positive control for endoglin and pSmad2. (D and E) Detection of phospho‐Smad2 in parental NIH/3T3 cells. Cells were treated with 20 μ g/mL of affinity‐purified LRG for 24 h and then treated with 2 ng/mL of TGF‐ β 1 for 30 min. Phospho‐Smad2, Smad2, LRG, and GAPDH were detected by western blot analysis. Band intensity of pSmad2 normalized to Smad2 is shown in Figure 5 E ( n = 3).

Techniques Used: Western Blot, Transfection, Incubation, Real-time Polymerase Chain Reaction, Expressing, Positive Control, Affinity Purification

Concentration of TGF ‐ β 1 in BALF and activation of Smad2 signaling in lungs from WT and LRG KO mice after bleomycin administration. (A) Comparison of TGF ‐ β levels in BALF between WT and LRG KO mice untreated (‐), and 7, 14 and 21 days after administration measured by ELISA ( n = 3–5 per group). (B) Phospho‐Smad2 and Smad2 levels in the lungs of WT mice and KO mice 21 days after bleomycin administration detected by western blot analysis. GAPDH was used as an internal control. (C) Quantification of pS mad2 band intensity normalized to Smad2 ( n = 5). Data were shown as mean ± SD . (D) Immunohistochemical analysis of α ‐ SMA in the lungs of WT and KO mice 21 days after administration. Scale bar = 200 μ m.
Figure Legend Snippet: Concentration of TGF ‐ β 1 in BALF and activation of Smad2 signaling in lungs from WT and LRG KO mice after bleomycin administration. (A) Comparison of TGF ‐ β levels in BALF between WT and LRG KO mice untreated (‐), and 7, 14 and 21 days after administration measured by ELISA ( n = 3–5 per group). (B) Phospho‐Smad2 and Smad2 levels in the lungs of WT mice and KO mice 21 days after bleomycin administration detected by western blot analysis. GAPDH was used as an internal control. (C) Quantification of pS mad2 band intensity normalized to Smad2 ( n = 5). Data were shown as mean ± SD . (D) Immunohistochemical analysis of α ‐ SMA in the lungs of WT and KO mice 21 days after administration. Scale bar = 200 μ m.

Techniques Used: Concentration Assay, Activation Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemistry

Effect of LRG on TGF ‐ β ‐induced Smad2 phosphorylation and downstream gene expression in L929. (A) Silver staining of affinity‐purified recombinant mouse LRG obtained from the culture supernatant of recombinant mouse LRG ‐expressing A549 cells using an anti‐ LRG antibody‐conjugated column. The sizes of molecular weight markers are indicated on the right lane. The size of the major band (indicated by arrowhead) corresponded to that of LRG (45–50 kD a). (B and C) Detection of phospho‐Smad2 by western blot analysis. L929 cells were treated with or without 20 μ g/ mL of LRG for 24 h. Cells were then stimulated with 2 ng/ mL of TGF ‐ β 1 for 0, 30, and 60 min. Anti‐phospho‐Smad2 (Ser465/467), anti‐Smad2, anti‐ LRG , and anti‐ GAPDH antibodies were used for the detection. Band intensity of pS mad2 normalized to Smad2 is shown in Figure 4 C ( n = 3). (D and E) Detection of phospho‐Smad2 by western blot analysis. L929 cells were treated without (lanes 1 and 2) or with 0.035 μ g/ mL (lane 3), 0.35 μ g/ mL (lane 4), and 3.5 μ g/ mL (lane 5) of LRG for 24 h and then with 2 ng/ mL of TGF ‐ β 1 for 30 min. Band intensity of pS mad2 normalized to Smad2 is shown in Figure 4 E ( n = 3). (F) Detection of LRG in culture supernatants of pc DNA ‐Lrg1 (containing full length LRG cDNA ) but not in those of pc DNA (vector without insertion). Supernatants from L929 cells after 24‐h culture in serum‐starved media were analyzed by western blot. (G and H) Detection of phospho‐Smad2 in pc DNA ‐Lrg1 and pc DNA cells after treatment with 2 ng/ mL of TGF ‐ β 1 for 0, 30, and 60 min. Band intensity of pS mad2 normalized to Smad2 is shown in Figure 4 H ( n = 3). (I) qPCR analysis of Serpine1 and Acta2 . L929 cells were treated with 2 ng/ mL of TGF ‐ β 1 for 6 h for Serpine1 or for 12 h for Acta2 . The relative expressions of Serpine1 and Acta2 , normalized to Hprt1 gene expression are shown.
Figure Legend Snippet: Effect of LRG on TGF ‐ β ‐induced Smad2 phosphorylation and downstream gene expression in L929. (A) Silver staining of affinity‐purified recombinant mouse LRG obtained from the culture supernatant of recombinant mouse LRG ‐expressing A549 cells using an anti‐ LRG antibody‐conjugated column. The sizes of molecular weight markers are indicated on the right lane. The size of the major band (indicated by arrowhead) corresponded to that of LRG (45–50 kD a). (B and C) Detection of phospho‐Smad2 by western blot analysis. L929 cells were treated with or without 20 μ g/ mL of LRG for 24 h. Cells were then stimulated with 2 ng/ mL of TGF ‐ β 1 for 0, 30, and 60 min. Anti‐phospho‐Smad2 (Ser465/467), anti‐Smad2, anti‐ LRG , and anti‐ GAPDH antibodies were used for the detection. Band intensity of pS mad2 normalized to Smad2 is shown in Figure 4 C ( n = 3). (D and E) Detection of phospho‐Smad2 by western blot analysis. L929 cells were treated without (lanes 1 and 2) or with 0.035 μ g/ mL (lane 3), 0.35 μ g/ mL (lane 4), and 3.5 μ g/ mL (lane 5) of LRG for 24 h and then with 2 ng/ mL of TGF ‐ β 1 for 30 min. Band intensity of pS mad2 normalized to Smad2 is shown in Figure 4 E ( n = 3). (F) Detection of LRG in culture supernatants of pc DNA ‐Lrg1 (containing full length LRG cDNA ) but not in those of pc DNA (vector without insertion). Supernatants from L929 cells after 24‐h culture in serum‐starved media were analyzed by western blot. (G and H) Detection of phospho‐Smad2 in pc DNA ‐Lrg1 and pc DNA cells after treatment with 2 ng/ mL of TGF ‐ β 1 for 0, 30, and 60 min. Band intensity of pS mad2 normalized to Smad2 is shown in Figure 4 H ( n = 3). (I) qPCR analysis of Serpine1 and Acta2 . L929 cells were treated with 2 ng/ mL of TGF ‐ β 1 for 6 h for Serpine1 or for 12 h for Acta2 . The relative expressions of Serpine1 and Acta2 , normalized to Hprt1 gene expression are shown.

Techniques Used: Expressing, Silver Staining, Affinity Purification, Recombinant, Molecular Weight, Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction

27) Product Images from "MicroRNAs Modulate Oxidative Stress in Hypertension through PARP-1 Regulation"

Article Title: MicroRNAs Modulate Oxidative Stress in Hypertension through PARP-1 Regulation

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2017/3984280

miR-103-2-5p and miR-585-5p decrease PARP-1 expression. HAECs (a, b) or HUVECs (c, d) were transfected with precursor mimics for miR-103-2-5p, miR-585-5p, or scrambled (Scr) control. After 48 hrs, cells were lysed for RNA and protein analysis. (a, c) miRNA and PARP1 mRNA levels were quantified by RT-qPCR analysis. (b, d) Lysates were analyzed by SDS-PAGE and immunoblotted with anti-PARP-1, anti-GAPDH, and anti- β -actin antibodies. Histograms represent the mean + SEM and from three independent experiments ∗ P
Figure Legend Snippet: miR-103-2-5p and miR-585-5p decrease PARP-1 expression. HAECs (a, b) or HUVECs (c, d) were transfected with precursor mimics for miR-103-2-5p, miR-585-5p, or scrambled (Scr) control. After 48 hrs, cells were lysed for RNA and protein analysis. (a, c) miRNA and PARP1 mRNA levels were quantified by RT-qPCR analysis. (b, d) Lysates were analyzed by SDS-PAGE and immunoblotted with anti-PARP-1, anti-GAPDH, and anti- β -actin antibodies. Histograms represent the mean + SEM and from three independent experiments ∗ P

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, SDS Page

28) Product Images from "SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability"

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00129-17

GFP-La expression and sumoylation in HEK293 cells. (A) Representative fluorescence images showing stable expression of GFP, GFP-La WT , and GFP-La SD in HEK293 cells. (B) Representative immunoblot of GFP-, GFP-La WT -, and GFP-La SD -expressing cells using anti-GFP antibody. GAPDH was used as a loading control. S-La, sumoylated La.
Figure Legend Snippet: GFP-La expression and sumoylation in HEK293 cells. (A) Representative fluorescence images showing stable expression of GFP, GFP-La WT , and GFP-La SD in HEK293 cells. (B) Representative immunoblot of GFP-, GFP-La WT -, and GFP-La SD -expressing cells using anti-GFP antibody. GAPDH was used as a loading control. S-La, sumoylated La.

Techniques Used: Expressing, Fluorescence

Sumoylation of La promotes cell proliferation via a STAT3-mediated mechanism. (A) Representative immunoblot of STAT3 in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. GAPDH was used as a loading control. (B) RT-qPCR analysis showing no significant difference in STAT3 mRNA levels in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. The values are normalized against GAPDH mRNA levels ( n = 3). (C) Representative immunoblot showing STAT3 protein levels in GFP-La WT - and GFP-La SD -expressing cells. GAPDH was used as a loading control. (D) Densitometry analysis showing significantly lower STAT3 protein expression in GFP-La SD -expressing cells than in GFP-La WT -expressing cells. The values are normalized against GAPDH protein levels ( n = 3). (E) Representative fluorescence images showing transient transfection of control (GFP) and RFP-STAT3 in cells transduced with lentiviral constructs expressing shC or sh5. The transfection efficiency was ∼30%. (F) Overexpression of STAT3 (RFP-STAT3) restored the numbers of La-depleted (sh5) cells ( n = 4; *, P = 0.0358). (G) Representative fluorescence images showing transient transfection of control and RFP-STAT3 in GFP-, GFP-La WT -, and GFP-La SD -expressing cells. The transfection efficiency was ∼30%. (H) Overexpression of STAT3 (RFP-STAT3) restored GFP-La SD cell numbers ( n = 3; *, P = 0.015). The asterisks indicate significant differences ( P
Figure Legend Snippet: Sumoylation of La promotes cell proliferation via a STAT3-mediated mechanism. (A) Representative immunoblot of STAT3 in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. GAPDH was used as a loading control. (B) RT-qPCR analysis showing no significant difference in STAT3 mRNA levels in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. The values are normalized against GAPDH mRNA levels ( n = 3). (C) Representative immunoblot showing STAT3 protein levels in GFP-La WT - and GFP-La SD -expressing cells. GAPDH was used as a loading control. (D) Densitometry analysis showing significantly lower STAT3 protein expression in GFP-La SD -expressing cells than in GFP-La WT -expressing cells. The values are normalized against GAPDH protein levels ( n = 3). (E) Representative fluorescence images showing transient transfection of control (GFP) and RFP-STAT3 in cells transduced with lentiviral constructs expressing shC or sh5. The transfection efficiency was ∼30%. (F) Overexpression of STAT3 (RFP-STAT3) restored the numbers of La-depleted (sh5) cells ( n = 4; *, P = 0.0358). (G) Representative fluorescence images showing transient transfection of control and RFP-STAT3 in GFP-, GFP-La WT -, and GFP-La SD -expressing cells. The transfection efficiency was ∼30%. (H) Overexpression of STAT3 (RFP-STAT3) restored GFP-La SD cell numbers ( n = 3; *, P = 0.015). The asterisks indicate significant differences ( P

Techniques Used: Transduction, Construct, Quantitative RT-PCR, Expressing, Fluorescence, Transfection, Over Expression

Sumoylation of La supports cell proliferation. (A) Representative immunoblot of La in HEK293 cells transduced with lentivirus expressing shC-, sh3-, and La shRNA5 (sh5)-specific shRNA constructs. GAPDH was used as a loading control. (B) Cell proliferation analysis of La-depleted cells as determined by the CyqQuant method. The La-depleted cells (sh5) show lower cell proliferation than control cells (shC) ( n = 3). (C) Cell proliferation analysis of GFP-La WT and GFP-La SD cells by the CyqQuant method. The GFP-La SD and GFP cells grew more slowly than GFP-La WT cells ( n = 3). (D) Cell cycle analysis of GFP, GFP-La WT , and GFP-La SD cells. The GFP and GFP-La SD cells showed significant accumulation in G 0 /G 1 phases, whereas GFP-La WT accumulated in the G 2 /M phases, ( n = 3). The asterisks indicate significant differences: *, P
Figure Legend Snippet: Sumoylation of La supports cell proliferation. (A) Representative immunoblot of La in HEK293 cells transduced with lentivirus expressing shC-, sh3-, and La shRNA5 (sh5)-specific shRNA constructs. GAPDH was used as a loading control. (B) Cell proliferation analysis of La-depleted cells as determined by the CyqQuant method. The La-depleted cells (sh5) show lower cell proliferation than control cells (shC) ( n = 3). (C) Cell proliferation analysis of GFP-La WT and GFP-La SD cells by the CyqQuant method. The GFP-La SD and GFP cells grew more slowly than GFP-La WT cells ( n = 3). (D) Cell cycle analysis of GFP, GFP-La WT , and GFP-La SD cells. The GFP and GFP-La SD cells showed significant accumulation in G 0 /G 1 phases, whereas GFP-La WT accumulated in the G 2 /M phases, ( n = 3). The asterisks indicate significant differences: *, P

Techniques Used: Transduction, Expressing, shRNA, Construct, Cell Cycle Assay

Sumoylation of La increases STAT3 mRNA binding by La. (A) RT-qPCR analysis showing no significant difference in STAT3 mRNA levels in GFP-La WT (Wt)- and GFP-La SD (K41/200R)-expressing cells. The values are normalized against GAPDH mRNA levels ( n = 3). (B) RT-PCR on RNA samples prepared from RIP experiments using HEK293 cells stably overexpressing GFP-La WT (Wt) or GFP-La SD (K41/200R). Significantly less STAT3 mRNA was associated with GFP-La SD than with GFP-La WT . The asterisks indicate a significant difference ( P
Figure Legend Snippet: Sumoylation of La increases STAT3 mRNA binding by La. (A) RT-qPCR analysis showing no significant difference in STAT3 mRNA levels in GFP-La WT (Wt)- and GFP-La SD (K41/200R)-expressing cells. The values are normalized against GAPDH mRNA levels ( n = 3). (B) RT-PCR on RNA samples prepared from RIP experiments using HEK293 cells stably overexpressing GFP-La WT (Wt) or GFP-La SD (K41/200R). Significantly less STAT3 mRNA was associated with GFP-La SD than with GFP-La WT . The asterisks indicate a significant difference ( P

Techniques Used: Binding Assay, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Stable Transfection

Sumoylation of La promotes STAT3 protein stability. (A) GFP-La WT - and GFP-La SD -expressing cells were treated with CHX (20 μM) for the indicated times and analyzed for STAT3 expression by immunoblot analysis. GAPDH protein levels were analyzed as a loading control. (B) Quantification of immunoblots revealed that STAT3 stability was reduced in GFP-La SD cells compared to GFP-La WT ( n = 3; *, P = 0.022 at 6 h and P = 0.023 at 12 h). The asterisks indicate significant differences ( P
Figure Legend Snippet: Sumoylation of La promotes STAT3 protein stability. (A) GFP-La WT - and GFP-La SD -expressing cells were treated with CHX (20 μM) for the indicated times and analyzed for STAT3 expression by immunoblot analysis. GAPDH protein levels were analyzed as a loading control. (B) Quantification of immunoblots revealed that STAT3 stability was reduced in GFP-La SD cells compared to GFP-La WT ( n = 3; *, P = 0.022 at 6 h and P = 0.023 at 12 h). The asterisks indicate significant differences ( P

Techniques Used: Expressing, Western Blot

29) Product Images from "ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons"

Article Title: ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.020099

Generation of FUS P525L iPSC lines by TALEN-directed mutagenesis. (A) Schematic of the TALEN/piggyBac combined strategy to generate the FUS P525L mutant iPSC lines. On the top, the WT FUS locus and TALENs are depicted. Below, the HDR donor construct is schematized. P PGK , phosphoglycerate kinase 1 promoter; PUΔTK, fusion between PuroR and DeltaTK (truncated version of HSV type 1 thymidine kinase). Yellow triangles represent enhanced piggyBac (ePB) terminal repeats. The P525L mutation is indicated in red. The expected product of the homologous recombination and the FUS locus after PB-mediated excision is shown at the bottom. Horizontal arrows indicate primers used for PCR amplification from genomic DNA of the fragments sequenced in B. (B) Sequencing results from WT I iPSCs (top), iPSCs modified by TALEN-directed HDR after removal of the selection cassette (middle, heterozygous; bottom, homozygous). The arrows indicate the targeted nucleotide in codon 525 (C, wild type; T, mutant). Further details are provided in supplementary material Figs S3-S6 . (C) Western blot analysis of FUS protein levels in iPSC lines used in this study. GAPDH is used as loading control. Densitometric quantification of FUS protein, relative to WT I, is shown below. (D) RT-qPCR analysis of FUS total mRNA and alternatively spliced mRNA devoid of exon 7 in iPSC lines. Quantification is relative to the levels in the WT I sample. (E) Immunostaining showing intracellular localization of WT and mutant FUS proteins in iPSCs. Scale bar: 10 μm. (F) Quantification of the immunostaining signal, showing FUS intracellular distribution in iPSC lines. In D and F, significant difference from the WT I was assessed by unpaired Student's t -test; * P
Figure Legend Snippet: Generation of FUS P525L iPSC lines by TALEN-directed mutagenesis. (A) Schematic of the TALEN/piggyBac combined strategy to generate the FUS P525L mutant iPSC lines. On the top, the WT FUS locus and TALENs are depicted. Below, the HDR donor construct is schematized. P PGK , phosphoglycerate kinase 1 promoter; PUΔTK, fusion between PuroR and DeltaTK (truncated version of HSV type 1 thymidine kinase). Yellow triangles represent enhanced piggyBac (ePB) terminal repeats. The P525L mutation is indicated in red. The expected product of the homologous recombination and the FUS locus after PB-mediated excision is shown at the bottom. Horizontal arrows indicate primers used for PCR amplification from genomic DNA of the fragments sequenced in B. (B) Sequencing results from WT I iPSCs (top), iPSCs modified by TALEN-directed HDR after removal of the selection cassette (middle, heterozygous; bottom, homozygous). The arrows indicate the targeted nucleotide in codon 525 (C, wild type; T, mutant). Further details are provided in supplementary material Figs S3-S6 . (C) Western blot analysis of FUS protein levels in iPSC lines used in this study. GAPDH is used as loading control. Densitometric quantification of FUS protein, relative to WT I, is shown below. (D) RT-qPCR analysis of FUS total mRNA and alternatively spliced mRNA devoid of exon 7 in iPSC lines. Quantification is relative to the levels in the WT I sample. (E) Immunostaining showing intracellular localization of WT and mutant FUS proteins in iPSCs. Scale bar: 10 μm. (F) Quantification of the immunostaining signal, showing FUS intracellular distribution in iPSC lines. In D and F, significant difference from the WT I was assessed by unpaired Student's t -test; * P

Techniques Used: Mutagenesis, TALENs, Construct, Homologous Recombination, Polymerase Chain Reaction, Amplification, Sequencing, Modification, Selection, Western Blot, Quantitative RT-PCR, Immunostaining

30) Product Images from "The genomic landscape of TERT promoter wildtype-IDH wildtype glioblastoma"

Article Title: The genomic landscape of TERT promoter wildtype-IDH wildtype glioblastoma

Journal: Nature Communications

doi: 10.1038/s41467-018-04448-6

Inactivating mutations in SMARCAL1 and ATRX , and rearrangements upstream of TERT are frequent in TERT p WT -IDH WT GBMs and related to distinct telomere maintenance mechanisms. a Based on ALT assessment by both telomere FISH and C-circle (dot blot), 38.5% (15/39) of TERT p WT -IDH WT GBMs exhibit signs of ALT. Of these, approximately half exhibit loss of ATRX expression (IHC) and half harbor mutations in SMARCAL1 , in a largely mutually exclusive manner. TERT rearrangements were identified by whole genome sequencing ( N = 8). Break-apart FISH was used to screen the cohort for TERT rearrangements, which were present in 50% (19/38) of all TERT p WT -IDH WT GBMs. b Circos plot of rearrangements identified upstream of TERT by whole genome sequencing of ALT-negative GBMs ( N = 8). Several cases were interchromosomal translocations (A, B, F), while the remaining cases were intrachromosomal (C, D, E). c The breakpoints of the rearrangements identified by whole genome sequencing span a region in the 50 kb upstream of TERT . d Examples of FISH on patient tumor tissue showing break-apart signal, indicating TERT- rearrangement. Arrows identify break-apart signals. e TERT expression was assessed by rt-qPCR relative to GAPDH . IDH WT -TERT SV ( n = 12) tumors exhibit significantly higher TERT expression than the IDH WT - ALT subgroup ( n = 9, P
Figure Legend Snippet: Inactivating mutations in SMARCAL1 and ATRX , and rearrangements upstream of TERT are frequent in TERT p WT -IDH WT GBMs and related to distinct telomere maintenance mechanisms. a Based on ALT assessment by both telomere FISH and C-circle (dot blot), 38.5% (15/39) of TERT p WT -IDH WT GBMs exhibit signs of ALT. Of these, approximately half exhibit loss of ATRX expression (IHC) and half harbor mutations in SMARCAL1 , in a largely mutually exclusive manner. TERT rearrangements were identified by whole genome sequencing ( N = 8). Break-apart FISH was used to screen the cohort for TERT rearrangements, which were present in 50% (19/38) of all TERT p WT -IDH WT GBMs. b Circos plot of rearrangements identified upstream of TERT by whole genome sequencing of ALT-negative GBMs ( N = 8). Several cases were interchromosomal translocations (A, B, F), while the remaining cases were intrachromosomal (C, D, E). c The breakpoints of the rearrangements identified by whole genome sequencing span a region in the 50 kb upstream of TERT . d Examples of FISH on patient tumor tissue showing break-apart signal, indicating TERT- rearrangement. Arrows identify break-apart signals. e TERT expression was assessed by rt-qPCR relative to GAPDH . IDH WT -TERT SV ( n = 12) tumors exhibit significantly higher TERT expression than the IDH WT - ALT subgroup ( n = 9, P

Techniques Used: Fluorescence In Situ Hybridization, Dot Blot, Expressing, Immunohistochemistry, Sequencing, Quantitative RT-PCR

31) Product Images from "Role of vasodilator-stimulated phosphoprotein in human cytomegalovirus-induced hyperpermeability of human endothelial cells"

Article Title: Role of vasodilator-stimulated phosphoprotein in human cytomegalovirus-induced hyperpermeability of human endothelial cells

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2018.6332

HCMV infection induces hyperpermeability through the Rac1/VASP signaling pathway in HUVEC-CRL-1730 cells. (A) Rac1 and VASP mRNA quantification, relative to GAPDH mRNA, was assessed by reverse transcription-polymerase chain reaction, in cells treated with siRNA-NC, siRNA-Rac1 or pflag-Rac1 for 48 h. Rac1 and VASP protein expression was determined and quantified by western blot analysis in cells treated with (B) siRNA-NC or siRNA-Rac1 and (C) pflag-CMV or pflag-Rac1 for 48 h. (D) The permeability of HUVEC-CRL-1730 cells, which were treated with siRNA-NC, siRNA-Rac1, pflag-CMV or the overexpression plasmid pflag-Rac1 for 24 h, was assessed using FITC-labeled dextran in a Transwell assay. After 24 h of HCMV infection, the permeability of HUVEC-CRL-1730 cells, which were treated with siRNA-NC and (E) siRNA-Rac1, pflag-CMV or the overexpression plasmid pflag-Rac1, or (F) siRNA-VASP, pEGFP-C1 or the overexpression plasmid pEGFP-VASP for 24 h, was assessed using FITC-labeled dextran in a Transwell assay. Data are expressed as the mean ± standard error of the mean (n=3 per group). *P
Figure Legend Snippet: HCMV infection induces hyperpermeability through the Rac1/VASP signaling pathway in HUVEC-CRL-1730 cells. (A) Rac1 and VASP mRNA quantification, relative to GAPDH mRNA, was assessed by reverse transcription-polymerase chain reaction, in cells treated with siRNA-NC, siRNA-Rac1 or pflag-Rac1 for 48 h. Rac1 and VASP protein expression was determined and quantified by western blot analysis in cells treated with (B) siRNA-NC or siRNA-Rac1 and (C) pflag-CMV or pflag-Rac1 for 48 h. (D) The permeability of HUVEC-CRL-1730 cells, which were treated with siRNA-NC, siRNA-Rac1, pflag-CMV or the overexpression plasmid pflag-Rac1 for 24 h, was assessed using FITC-labeled dextran in a Transwell assay. After 24 h of HCMV infection, the permeability of HUVEC-CRL-1730 cells, which were treated with siRNA-NC and (E) siRNA-Rac1, pflag-CMV or the overexpression plasmid pflag-Rac1, or (F) siRNA-VASP, pEGFP-C1 or the overexpression plasmid pEGFP-VASP for 24 h, was assessed using FITC-labeled dextran in a Transwell assay. Data are expressed as the mean ± standard error of the mean (n=3 per group). *P

Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Permeability, Over Expression, Plasmid Preparation, Labeling, Transwell Assay

32) Product Images from "ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis"

Article Title: ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-018-0992-0

Expression of ETV4 and MMP13 in TAC-Ctrl/ETV4 and MMT-Ctrl/ETV4 cells. a and b Relative ETV4 ( a ) or MMP13 ( b ) mRNA expression in TAC/MMT-Ctrl and TAC/MMT-ETV4-overexpressing cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in TAC/MMT-Ctrl cells was arbitrarily = 1. Error bars indicate SD. **** P ≤ 0.0001; ** P ≤ 0.01. c and d Western blot analysis of ETV4 protein expression (61 kDa) ( c ) or MMP13 protein expression (60 kDa) ( d ) in TAC/MMT-Ctrl and TAC/MMT-ETV4 cells. GAPDH expression served as the loading control. e Western blot analysis of the secreted MMP13 protein expression (55 kDa) from the supernatant of MMT-Ctrl and MMT-ETV4-overexpressing cells. f Zymographic analysis of MMP13 protein activity (55 kDa) in MMT-Ctrl and MMT-ETV4 cells
Figure Legend Snippet: Expression of ETV4 and MMP13 in TAC-Ctrl/ETV4 and MMT-Ctrl/ETV4 cells. a and b Relative ETV4 ( a ) or MMP13 ( b ) mRNA expression in TAC/MMT-Ctrl and TAC/MMT-ETV4-overexpressing cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in TAC/MMT-Ctrl cells was arbitrarily = 1. Error bars indicate SD. **** P ≤ 0.0001; ** P ≤ 0.01. c and d Western blot analysis of ETV4 protein expression (61 kDa) ( c ) or MMP13 protein expression (60 kDa) ( d ) in TAC/MMT-Ctrl and TAC/MMT-ETV4 cells. GAPDH expression served as the loading control. e Western blot analysis of the secreted MMP13 protein expression (55 kDa) from the supernatant of MMT-Ctrl and MMT-ETV4-overexpressing cells. f Zymographic analysis of MMP13 protein activity (55 kDa) in MMT-Ctrl and MMT-ETV4 cells

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay

33) Product Images from "Extracts of Artocarpus communis Induce Mitochondria-Associated Apoptosis via Pro-oxidative Activity in Human Glioblastoma Cells"

Article Title: Extracts of Artocarpus communis Induce Mitochondria-Associated Apoptosis via Pro-oxidative Activity in Human Glioblastoma Cells

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00411

Artocarpin induces apoptosis via the mitochondrial pathway. (A) Cells were treated with various artocarpin concentrations for 24 h. Effect of artocarpin on U87 mitochondrial depolarization was measured with JC-1 staining and a fluorescence plate reader. (B) Cells were treated with artocarpin for 24 h then the Bad, Bax, and Bcl-2 protein expression levels were determined by western blot. (C) Cells were treated with artocarpin for 16- or 24 h. Cytosolic- and mitochondrial fractions were prepared and subjected to western blot with anti-cytochrome c antibody. GAPDH was used as a marker protein for cytosolic fractions. COX IV was used as a marker protein for mitochondrial fractions. (D) Cells were pretreated with MCI-186 (10 μM) for 1 h then treated with artocarpin for 24 h. Bad, Bax, and Bcl-2 protein expression levels were determined by western blot. (E) Cells were pretreated with MCI-186 (10 μM) for 1 h then treated with artocarpin for 24 h. Cytosolic- and mitochondrial fractions were prepared and subjected to western blot with anti-cytochrome c antibody. Data are expressed as means ± SE of three independent experiments. # P
Figure Legend Snippet: Artocarpin induces apoptosis via the mitochondrial pathway. (A) Cells were treated with various artocarpin concentrations for 24 h. Effect of artocarpin on U87 mitochondrial depolarization was measured with JC-1 staining and a fluorescence plate reader. (B) Cells were treated with artocarpin for 24 h then the Bad, Bax, and Bcl-2 protein expression levels were determined by western blot. (C) Cells were treated with artocarpin for 16- or 24 h. Cytosolic- and mitochondrial fractions were prepared and subjected to western blot with anti-cytochrome c antibody. GAPDH was used as a marker protein for cytosolic fractions. COX IV was used as a marker protein for mitochondrial fractions. (D) Cells were pretreated with MCI-186 (10 μM) for 1 h then treated with artocarpin for 24 h. Bad, Bax, and Bcl-2 protein expression levels were determined by western blot. (E) Cells were pretreated with MCI-186 (10 μM) for 1 h then treated with artocarpin for 24 h. Cytosolic- and mitochondrial fractions were prepared and subjected to western blot with anti-cytochrome c antibody. Data are expressed as means ± SE of three independent experiments. # P

Techniques Used: Staining, Fluorescence, Expressing, Western Blot, Marker

34) Product Images from "Short-form Ron is a novel determinant of ovarian cancer initiation and progression"

Article Title: Short-form Ron is a novel determinant of ovarian cancer initiation and progression

Journal: Genes & Cancer

doi: 10.18632/genesandcancer.109

The expression and activity of Ron receptor isoforms in primary tumors and high-grade serous ovarian (HG-SOC) PDXs A. Upper panel displays western blot analysis of tumor lysates from patients assayed for phosphorylated (active) sfRon and Ron. Lower panel shows western blot analysis of total levels of Ron isoforms. The higher molecular weight sfRon bands (sfRon-HMW), which are putative, posttranslationally modified sfRon forms were also noted. The blots were stripped and re-probed for β-actin. Lanes represent: healthy ovary (1, 2, 3); ovarian adenocarcinoma (4); carcinosarcoma (5); endometrioid adenocarcinoma (6) and HG-SOC (7). B. Upper panel displays western blot analysis of tumor lysates from HG-SOC PDXs assayed for phosphorylated Ron isoforms. Lower panel shows western blot analysis of total levels of Ron isoforms. The blots were stripped and re-probed for β-actin or GAPDH. C. The expression of Ron isoforms was assessed by WES capillary electrophoresis-based protein assay in OVCAR3-sfRon cell line engineered to express sfRon vs. parental OVACR3 cells and compared with sfRon positive or negative HG-SOC PDXs. GAPDH was used as loading control.
Figure Legend Snippet: The expression and activity of Ron receptor isoforms in primary tumors and high-grade serous ovarian (HG-SOC) PDXs A. Upper panel displays western blot analysis of tumor lysates from patients assayed for phosphorylated (active) sfRon and Ron. Lower panel shows western blot analysis of total levels of Ron isoforms. The higher molecular weight sfRon bands (sfRon-HMW), which are putative, posttranslationally modified sfRon forms were also noted. The blots were stripped and re-probed for β-actin. Lanes represent: healthy ovary (1, 2, 3); ovarian adenocarcinoma (4); carcinosarcoma (5); endometrioid adenocarcinoma (6) and HG-SOC (7). B. Upper panel displays western blot analysis of tumor lysates from HG-SOC PDXs assayed for phosphorylated Ron isoforms. Lower panel shows western blot analysis of total levels of Ron isoforms. The blots were stripped and re-probed for β-actin or GAPDH. C. The expression of Ron isoforms was assessed by WES capillary electrophoresis-based protein assay in OVCAR3-sfRon cell line engineered to express sfRon vs. parental OVACR3 cells and compared with sfRon positive or negative HG-SOC PDXs. GAPDH was used as loading control.

Techniques Used: Expressing, Activity Assay, Western Blot, Molecular Weight, Modification, Electrophoresis

The sfRon signaling pathway in ovarian cancer A. Analysis of the effects of sfRon expression on the activity of PI3K and MAPK signaling pathways downstream from sfRon. Indicated proteins were detected by WES capillary electrophoresis-based protein assay (sfRon, GAPDH, pAKT Thr308 , pPDK1, PDK1) or standard Western Blot (pAKT Ser473 , pERK, panAKT, panERK). Whole Blots or WES images are shown in Supplementary Fig. 1 . B. Analysis of the activity of TGFβ pathway and the expression of EMT marker proteins in OVCAR3-sfRon vs parental OVCAR3 cells. Indicated proteins were detected by standard Western Blot (TGFβ1, pSMAD2/3, SMAD2/3) or by WES capillary electrophoresis-based protein assay (E-cadherin, N-cadherin, vimentin). Whole Blots or WES images are shown in Supplementary Fig. 2 . C. The qRT-PCR analysis of EMT related transcription factors such as SIP1, SLUG, SNAIL and ZEB1.
Figure Legend Snippet: The sfRon signaling pathway in ovarian cancer A. Analysis of the effects of sfRon expression on the activity of PI3K and MAPK signaling pathways downstream from sfRon. Indicated proteins were detected by WES capillary electrophoresis-based protein assay (sfRon, GAPDH, pAKT Thr308 , pPDK1, PDK1) or standard Western Blot (pAKT Ser473 , pERK, panAKT, panERK). Whole Blots or WES images are shown in Supplementary Fig. 1 . B. Analysis of the activity of TGFβ pathway and the expression of EMT marker proteins in OVCAR3-sfRon vs parental OVCAR3 cells. Indicated proteins were detected by standard Western Blot (TGFβ1, pSMAD2/3, SMAD2/3) or by WES capillary electrophoresis-based protein assay (E-cadherin, N-cadherin, vimentin). Whole Blots or WES images are shown in Supplementary Fig. 2 . C. The qRT-PCR analysis of EMT related transcription factors such as SIP1, SLUG, SNAIL and ZEB1.

Techniques Used: Expressing, Activity Assay, Electrophoresis, Western Blot, Marker, Quantitative RT-PCR

35) Product Images from "Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway"

Article Title: Sirtuin 3 enhanced drug sensitivity of human hepatoma cells through glutathione S-transferase pi 1/JNK signaling pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.10319

SIRT3 overexpression regulated GSTP1/JNK signaling pathway A. SIRT3 inhibited GSTP1 mRNA level. Forty-eight hour after lentivirus infection, SMMC-7721, Huh-7 and PLC/PRF/5 cells were treated with doxorubicin (1 μg/ml), cisplatin (1 μg/ml) or epirubicin (0.5 μg/ml) for 48 h. The mRNA level of GSTP1 was detected by qPCR. β-actin mRNA expression was used as an internal control. B. SIRT3 inhibited GSTP1 protein level. The protein level of GSTP1 in SMMC-7721, Huh-7 and PLC/PRF/5 cells with or without chemotherapeutic agents was determined by western blot analysis. GAPDH was used as a loading control. C-E. The expression of total JNK, total c-Jun, phosphorylated-JNK, phosphorylated-c-Jun and Bim were examined in SMMC-7721 (C), Huh-7 (D) and PLC/PRF/5 (E) cells treated with doxorubicin (1 μg/ml), cisplatin (1 μg/ml) or epirubicin (0.5 μg/ml). GAPDH was used as a loading control. F. SIRT3 decreased the amount of GSTP1 that was associated with JNK. Immunoprecipitation was conducted with lysates prepared from SIRT3-overexpressing HCC cells (SMMC-7721 and Huh-7) or control cells treated with doxorubicin (1 μg/ml) by anti-JNK antibody, and immunoblotting was performed with anti-GSTP1 antibody.
Figure Legend Snippet: SIRT3 overexpression regulated GSTP1/JNK signaling pathway A. SIRT3 inhibited GSTP1 mRNA level. Forty-eight hour after lentivirus infection, SMMC-7721, Huh-7 and PLC/PRF/5 cells were treated with doxorubicin (1 μg/ml), cisplatin (1 μg/ml) or epirubicin (0.5 μg/ml) for 48 h. The mRNA level of GSTP1 was detected by qPCR. β-actin mRNA expression was used as an internal control. B. SIRT3 inhibited GSTP1 protein level. The protein level of GSTP1 in SMMC-7721, Huh-7 and PLC/PRF/5 cells with or without chemotherapeutic agents was determined by western blot analysis. GAPDH was used as a loading control. C-E. The expression of total JNK, total c-Jun, phosphorylated-JNK, phosphorylated-c-Jun and Bim were examined in SMMC-7721 (C), Huh-7 (D) and PLC/PRF/5 (E) cells treated with doxorubicin (1 μg/ml), cisplatin (1 μg/ml) or epirubicin (0.5 μg/ml). GAPDH was used as a loading control. F. SIRT3 decreased the amount of GSTP1 that was associated with JNK. Immunoprecipitation was conducted with lysates prepared from SIRT3-overexpressing HCC cells (SMMC-7721 and Huh-7) or control cells treated with doxorubicin (1 μg/ml) by anti-JNK antibody, and immunoblotting was performed with anti-GSTP1 antibody.

Techniques Used: Over Expression, Infection, Planar Chromatography, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunoprecipitation

36) Product Images from "Antibody neutralization of cell-surface gC1qR/HABP1/SF2-p32 prevents lamellipodia formation and tumorigenesis"

Article Title: Antibody neutralization of cell-surface gC1qR/HABP1/SF2-p32 prevents lamellipodia formation and tumorigenesis

Journal: Oncotarget

doi: 10.18632/oncotarget.10267

Antibody neutralization of gC1qR prevents the activation of focal adhesion kinase (FAK) A. A549 cells were grown after plating in the presence of 10 μg/mL of mock IgG or mAb 3D9. Cellular morphology was observed with a light microscopy 6 h and 48 h after cell plating. Scale bar = 50 μm. B and C. A549 was serum-starved for 18 h and pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated for 10 min by FBS (10%), EGF (50 ng/mL), IGF-1 (100 ng/mL) and PDGF (20ng/mL) for 10 min. Phosphorylated (at Y397 or Y925) and total forms of FAK were analyzed by immunoblotting using GAPDH as a loading control (B and C). The ratio of p-FAK/FAK D and E. was calculated from the band intensities for p-FAK (Y397), p-FAK (Y925) and FAK in the immunoblottings (B and C). Cellular localization of p-FAK (Y397) and FAK was determined by immunofluorescence F. Scale bar = 10 μm.
Figure Legend Snippet: Antibody neutralization of gC1qR prevents the activation of focal adhesion kinase (FAK) A. A549 cells were grown after plating in the presence of 10 μg/mL of mock IgG or mAb 3D9. Cellular morphology was observed with a light microscopy 6 h and 48 h after cell plating. Scale bar = 50 μm. B and C. A549 was serum-starved for 18 h and pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated for 10 min by FBS (10%), EGF (50 ng/mL), IGF-1 (100 ng/mL) and PDGF (20ng/mL) for 10 min. Phosphorylated (at Y397 or Y925) and total forms of FAK were analyzed by immunoblotting using GAPDH as a loading control (B and C). The ratio of p-FAK/FAK D and E. was calculated from the band intensities for p-FAK (Y397), p-FAK (Y925) and FAK in the immunoblottings (B and C). Cellular localization of p-FAK (Y397) and FAK was determined by immunofluorescence F. Scale bar = 10 μm.

Techniques Used: Neutralization, Activation Assay, Light Microscopy, Immunofluorescence

Antibody neutralization of gC1qR prevents receptor tyrosine kinases (RTKs) signaling A-D. A549 cells were serum-starved for 18 h and pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated for 10 min by FBS (10%) (A), EGF (50 ng/mL) (B), IGF-1 (100 ng/mL) (C) and PDGF (20 ng/mL) (D) for 10 min. H-J. MDA-MB-231 cells were serum-starved for 18 h and pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated for 10 min by FBS (10%) (H), EGF (50 ng/mL) (I) and IGF-1 (100 ng/mL) (J). Phosphorylated and total forms of Akt, Erk1/2, EGFR, IGFR and PDGFR were analyzed by immunoblotting using GAPDH as a loading control. The ratio of p-Akt/Akt E and K. , p-Erk/Erk F and L. , p-EGFR/EGFR G and M. , p-IGFR/IGFR (G and M) and p-PDGFR/PDGFR (G) was calculated from the band intensities for p-Akt, Akt, p-Erk1/2, Erk1/2, p-EGFR, EGFR, p-IGFR, IGFR, p-PDGFR and PDGFR in the immunoblottings (A-D and H-J).
Figure Legend Snippet: Antibody neutralization of gC1qR prevents receptor tyrosine kinases (RTKs) signaling A-D. A549 cells were serum-starved for 18 h and pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated for 10 min by FBS (10%) (A), EGF (50 ng/mL) (B), IGF-1 (100 ng/mL) (C) and PDGF (20 ng/mL) (D) for 10 min. H-J. MDA-MB-231 cells were serum-starved for 18 h and pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated for 10 min by FBS (10%) (H), EGF (50 ng/mL) (I) and IGF-1 (100 ng/mL) (J). Phosphorylated and total forms of Akt, Erk1/2, EGFR, IGFR and PDGFR were analyzed by immunoblotting using GAPDH as a loading control. The ratio of p-Akt/Akt E and K. , p-Erk/Erk F and L. , p-EGFR/EGFR G and M. , p-IGFR/IGFR (G and M) and p-PDGFR/PDGFR (G) was calculated from the band intensities for p-Akt, Akt, p-Erk1/2, Erk1/2, p-EGFR, EGFR, p-IGFR, IGFR, p-PDGFR and PDGFR in the immunoblottings (A-D and H-J).

Techniques Used: Neutralization, Multiple Displacement Amplification

Antibody neutralization of gC1qR prevents lamellipodia formation, cell migration and VEGF signaling in HUVEC A and B. Human umbilical vein endothelial cells (HUVEC) were grown to non-confluency, serum-starved with 0.2% FBS-containing endothelial basal media (EBM-2) for 18 h, pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated with endothelial growth media (EGM-2) (A) or vascular endothelial growth factor (VEGF, 50 ng/mL) (B) for 10 min. Cellular localization of gC1qR and CD44 was determined by non-permeabilized immunofluorescence (right panels). Lamellipodia-containing cells (%) were statistically determined from 12 different imaging fields (left panels). Bar = 20 μm. C. EGM-2-induced cell migration of HUVEC was determined by trans-well assay in the presence of 10 μg/mL of mock IgG or mAb 3D9. The trans-well membrane was stained by crystal violet (left panel). Cell migration was statistically determined from three independent experiments (right panel). Bar = 50 μm. D. Serum-starved HUVEC were serum-starved in 0.2% FBS-containing EBM-2 for 18 h and trypsinized. The cells were loaded into a Matrigel-coated cell plate and tube formation was induced with EGM-2 for 18 h in the presence of 200 μg/mL mock IgG or mAb 3D9. Scale bar = 100 μm. E and F. Serum-starved HUVEC were pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated by EGM-2 (E) or VEGF (50 ng/mL) (F) for 10 min. Phosphorylated and total forms of Akt, Erk1/2 and VEGFR were analyzed by immunoblotting using GAPDH as a loading control. * p
Figure Legend Snippet: Antibody neutralization of gC1qR prevents lamellipodia formation, cell migration and VEGF signaling in HUVEC A and B. Human umbilical vein endothelial cells (HUVEC) were grown to non-confluency, serum-starved with 0.2% FBS-containing endothelial basal media (EBM-2) for 18 h, pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated with endothelial growth media (EGM-2) (A) or vascular endothelial growth factor (VEGF, 50 ng/mL) (B) for 10 min. Cellular localization of gC1qR and CD44 was determined by non-permeabilized immunofluorescence (right panels). Lamellipodia-containing cells (%) were statistically determined from 12 different imaging fields (left panels). Bar = 20 μm. C. EGM-2-induced cell migration of HUVEC was determined by trans-well assay in the presence of 10 μg/mL of mock IgG or mAb 3D9. The trans-well membrane was stained by crystal violet (left panel). Cell migration was statistically determined from three independent experiments (right panel). Bar = 50 μm. D. Serum-starved HUVEC were serum-starved in 0.2% FBS-containing EBM-2 for 18 h and trypsinized. The cells were loaded into a Matrigel-coated cell plate and tube formation was induced with EGM-2 for 18 h in the presence of 200 μg/mL mock IgG or mAb 3D9. Scale bar = 100 μm. E and F. Serum-starved HUVEC were pretreated with 10 μg/mL of mock IgG or mAb 3D9 for 4 h and stimulated by EGM-2 (E) or VEGF (50 ng/mL) (F) for 10 min. Phosphorylated and total forms of Akt, Erk1/2 and VEGFR were analyzed by immunoblotting using GAPDH as a loading control. * p

Techniques Used: Neutralization, Migration, Immunofluorescence, Imaging, Staining

37) Product Images from "CHTM1, a novel metabolic marker deregulated in human malignancies"

Article Title: CHTM1, a novel metabolic marker deregulated in human malignancies

Journal: Oncogene

doi: 10.1038/s41388-017-0051-9

CHTM1 regulates lipid metabolism. Cells were cultured in the Lab-tek II chamber slides and subjected to glucose/glutamine deprivation. Cells were then fixed with 4% paraformaldehyde for 30 minutes. Fixed cells were stained with 0.1 mg/ml BODIPY for 10 minutes and washed twice with PBS. DAPI was used for nuclear staining and slides were analyzed using a Zeiss LSM 780 confocal microscope that had the required filters. Images were processed using Image J software. (A) CHTM1 knockdown causes increase in cellular lipid content. Representative confocal images of scrambled and CHTM1 knockdown MCF-7 breast cancer cells (Olympus AX70, Objective 60X). and UACC-62 melanoma cells stained with BODIPY and DAPI are shown (Zeiss LSM 780). Scale bar represents 20 μm. Left panel, scrambled and CHTM1 knockdown MCF-7 breast cancer cells grown in complete medium. Right Panel, scrambled and CHTM1 knockdown UACC-62, melanoma cells grown in complete medium or glucose/glutamine (Glc/Gln)-deprived medium for 24h (B) Oil red O (ORO) staining in scramble and CHTM1 knockdown MCF-7 cells grown in complete medium or in Glc/Gln-deprived medium for 24h. ORO staining shows increased lipid content in CHTM1 knockdown cells. (C) Left panel, representative confocal images of control and CHTM1 overexpressing MCF-7 cells grown in Glc/Gln-deprived medium for 24h (Zeiss LSM 780). Scale bar represents 20 μm. Right panel: Oil red O staining in CHTM1 overexpressing MCF-7 cells grown in Glc/Gln-deprived conditions for 24h show decrease in lipid content. (D) CHTM1 knockdown decreases mRNA levels of genes involved in fatty acid oxidation (CPT1b, MCAD AND PPARα). (E) CHTM1 knockdown decreases mRNA levels of genes involved in fatty acid synthesis (ACC1, FAS, SREBP1c, PPARγ). mRNA levels were analyzed by Q-PCR and the values represent SEM± of three independent experiments. Two-step quantitative real-time PCR was performed using the iScript cDNA Synthesis Kit and iQ SYBR Green super mix from Bio-Rad (Hercules, CA, USA). Specific gene values were normalized to the C(T) values of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA within the same sample. The following primer sets were used: ACC1 forward: 5′-ATC CCG TAC CTT CTT CTA CTG -3′; reverse: 5′-CCC AAA CAT AAG CCT TCA CTG -3′; FAS forward: 5′-CAG GGA CAA CCT GGA GTT CT -3′; reverse: 5′-CTG TGG TCC CAC TTG ATG AGT -3′; SREBP1c forward: 5′-GGA GGG GTA GGG CCA ACG GCC T -3′; reverse: 5′-CAT GTC TTC GAA AGT GCA ATC C -3′; PPAR′ forward: 5′-GGC TTC ATG ACA AGG GAG TTT C -3′; reverse: 5′-AAC TCA AAC TTG GGC TCC ATA AAG -3′; CHTM1 forward: 5′-GAG CAG TAT GGC CAG TGT GT -3′; reverse: 5′-ACT GGG CAA TGC TCA TCT TA -3′; MCAD forward: 5′-TAC TTG TAG AGC ACC AAG CAA TAT CA -3′; reverse: 5′-TGC TCT CTG GTA ACT CAT TCT AGC TAG T -3′; PPARα forward: 5′-GGC GAG GAT AGT TCT GGA AGC -3′; reverse: 5′-CAC AGG ATA AGT CAC CGA GGA G -3′; PGC-1α forward: 5′-TGC CCT GGA TTG TTG ACA TGA -3′; reverse: 5′-TTT GTC AGG CTG GGG GTA GG -3′, CPT1b: forward: GCGCTGGAGGTGGCTTT, reverse: TCGTGTTCTCGCCTGCAAT; *p
Figure Legend Snippet: CHTM1 regulates lipid metabolism. Cells were cultured in the Lab-tek II chamber slides and subjected to glucose/glutamine deprivation. Cells were then fixed with 4% paraformaldehyde for 30 minutes. Fixed cells were stained with 0.1 mg/ml BODIPY for 10 minutes and washed twice with PBS. DAPI was used for nuclear staining and slides were analyzed using a Zeiss LSM 780 confocal microscope that had the required filters. Images were processed using Image J software. (A) CHTM1 knockdown causes increase in cellular lipid content. Representative confocal images of scrambled and CHTM1 knockdown MCF-7 breast cancer cells (Olympus AX70, Objective 60X). and UACC-62 melanoma cells stained with BODIPY and DAPI are shown (Zeiss LSM 780). Scale bar represents 20 μm. Left panel, scrambled and CHTM1 knockdown MCF-7 breast cancer cells grown in complete medium. Right Panel, scrambled and CHTM1 knockdown UACC-62, melanoma cells grown in complete medium or glucose/glutamine (Glc/Gln)-deprived medium for 24h (B) Oil red O (ORO) staining in scramble and CHTM1 knockdown MCF-7 cells grown in complete medium or in Glc/Gln-deprived medium for 24h. ORO staining shows increased lipid content in CHTM1 knockdown cells. (C) Left panel, representative confocal images of control and CHTM1 overexpressing MCF-7 cells grown in Glc/Gln-deprived medium for 24h (Zeiss LSM 780). Scale bar represents 20 μm. Right panel: Oil red O staining in CHTM1 overexpressing MCF-7 cells grown in Glc/Gln-deprived conditions for 24h show decrease in lipid content. (D) CHTM1 knockdown decreases mRNA levels of genes involved in fatty acid oxidation (CPT1b, MCAD AND PPARα). (E) CHTM1 knockdown decreases mRNA levels of genes involved in fatty acid synthesis (ACC1, FAS, SREBP1c, PPARγ). mRNA levels were analyzed by Q-PCR and the values represent SEM± of three independent experiments. Two-step quantitative real-time PCR was performed using the iScript cDNA Synthesis Kit and iQ SYBR Green super mix from Bio-Rad (Hercules, CA, USA). Specific gene values were normalized to the C(T) values of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) mRNA within the same sample. The following primer sets were used: ACC1 forward: 5′-ATC CCG TAC CTT CTT CTA CTG -3′; reverse: 5′-CCC AAA CAT AAG CCT TCA CTG -3′; FAS forward: 5′-CAG GGA CAA CCT GGA GTT CT -3′; reverse: 5′-CTG TGG TCC CAC TTG ATG AGT -3′; SREBP1c forward: 5′-GGA GGG GTA GGG CCA ACG GCC T -3′; reverse: 5′-CAT GTC TTC GAA AGT GCA ATC C -3′; PPAR′ forward: 5′-GGC TTC ATG ACA AGG GAG TTT C -3′; reverse: 5′-AAC TCA AAC TTG GGC TCC ATA AAG -3′; CHTM1 forward: 5′-GAG CAG TAT GGC CAG TGT GT -3′; reverse: 5′-ACT GGG CAA TGC TCA TCT TA -3′; MCAD forward: 5′-TAC TTG TAG AGC ACC AAG CAA TAT CA -3′; reverse: 5′-TGC TCT CTG GTA ACT CAT TCT AGC TAG T -3′; PPARα forward: 5′-GGC GAG GAT AGT TCT GGA AGC -3′; reverse: 5′-CAC AGG ATA AGT CAC CGA GGA G -3′; PGC-1α forward: 5′-TGC CCT GGA TTG TTG ACA TGA -3′; reverse: 5′-TTT GTC AGG CTG GGG GTA GG -3′, CPT1b: forward: GCGCTGGAGGTGGCTTT, reverse: TCGTGTTCTCGCCTGCAAT; *p

Techniques Used: Cell Culture, Staining, Microscopy, Software, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, SYBR Green Assay, CTG Assay, Countercurrent Chromatography, Cellular Antioxidant Activity Assay, Activated Clotting Time Assay, Pyrolysis Gas Chromatography

38) Product Images from "Estrogen Receptor α Is Crucial in Zearalenone-Induced Invasion and Migration of Prostate Cancer Cells"

Article Title: Estrogen Receptor α Is Crucial in Zearalenone-Induced Invasion and Migration of Prostate Cancer Cells

Journal: Toxins

doi: 10.3390/toxins10030098

ZEA induces PC3 cell invasion dependent on ERα. ( A ) the results of ICC of ERα and ERβ (red stained) and DAPI (nuclei staining in blue); ( B ) the results from the cell invasion assay (modified Boyden chamber) are expressed as mean ± SE and presented as % of control; ( C ) representative results from cell invasion experiment, cells were stained with crystal violet and photographed in inverted microscopy; ( D , E ) the results from zymography assay are expressed as mean ± SE value as % of control cells; ( F ) representative results from zymography assay; ( G , H ) the results from the RT-qPCR study are expressed as mean ± SE and relative expression of genes was calculated as a ratio of ΔΔCt calculated expression of the gene od interest and reference genes: H3F3A , RPLP0 and RPS17 ; ( I ) the results from Western blot conducted to evaluate the expression of MMP-2, GAPDH was used as a reference. Statistically significant results were marked with lines, * p
Figure Legend Snippet: ZEA induces PC3 cell invasion dependent on ERα. ( A ) the results of ICC of ERα and ERβ (red stained) and DAPI (nuclei staining in blue); ( B ) the results from the cell invasion assay (modified Boyden chamber) are expressed as mean ± SE and presented as % of control; ( C ) representative results from cell invasion experiment, cells were stained with crystal violet and photographed in inverted microscopy; ( D , E ) the results from zymography assay are expressed as mean ± SE value as % of control cells; ( F ) representative results from zymography assay; ( G , H ) the results from the RT-qPCR study are expressed as mean ± SE and relative expression of genes was calculated as a ratio of ΔΔCt calculated expression of the gene od interest and reference genes: H3F3A , RPLP0 and RPS17 ; ( I ) the results from Western blot conducted to evaluate the expression of MMP-2, GAPDH was used as a reference. Statistically significant results were marked with lines, * p

Techniques Used: Immunocytochemistry, Staining, Invasion Assay, Modification, Inverted Microscopy, Zymography, Quantitative RT-PCR, Expressing, Western Blot

39) Product Images from "The Transition from Proliferation to Differentiation in Colorectal Cancer Is Regulated by the Calcium Activated Chloride Channel A1"

Article Title: The Transition from Proliferation to Differentiation in Colorectal Cancer Is Regulated by the Calcium Activated Chloride Channel A1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060861

Expression of CLCA1, ALPI and sucrase-isomaltase was upregulated during spontaneous differentiation of Caco-2 monolayer. A and B. Expression of CLCA1 subunits (38 KD and 90 KD) were up-regulated after 24 hour of confluent culture and reached a peak at 10 days of culture. C. ALPI as a marker of Caco-2 cell differentiation was up-regulated significantly after 4 days of confluent culture. D. Expression of sucrase-isomaltase (SI), another cell differentiation marker, also was increased significantly after 4 days of confluent culture. E. Expression of β-catenin was enhanced slightly over time in culture. The histograms in A to E show the relative intensity of CLCA1, ALPI, SI and β-catenin expressed as a ratio with respect to the GAPDH control. All results were analyzed from three independent experiments.
Figure Legend Snippet: Expression of CLCA1, ALPI and sucrase-isomaltase was upregulated during spontaneous differentiation of Caco-2 monolayer. A and B. Expression of CLCA1 subunits (38 KD and 90 KD) were up-regulated after 24 hour of confluent culture and reached a peak at 10 days of culture. C. ALPI as a marker of Caco-2 cell differentiation was up-regulated significantly after 4 days of confluent culture. D. Expression of sucrase-isomaltase (SI), another cell differentiation marker, also was increased significantly after 4 days of confluent culture. E. Expression of β-catenin was enhanced slightly over time in culture. The histograms in A to E show the relative intensity of CLCA1, ALPI, SI and β-catenin expressed as a ratio with respect to the GAPDH control. All results were analyzed from three independent experiments.

Techniques Used: Expressing, Marker, Cell Differentiation

CLCA1 is required for spontaneous differentiation in Caco-2 cells. A. Caco-2 cells were transfected transiently with 0, 50, 100, 150 and 200 nM siRNA clca1 and blotted for CLCA1. siRNA clca1 at 100 nM or above effectively inhibited CLCA1 and downregulated expression of ALPI and β-catenin. B. Immunofluorescent staining showed the expression of β-catenin in confluent cultures of Caco-2 cells. β-catenin was located mainly in the nucleus of the cells at early stages of culture (2 days). After 10 days culture, β-catenin had translocated to the cell membrane. Knockdown of CLCA1 reduced distribution of β-catenin on the membrane. C. Caco-2 cells were treated with either siRNA negative control or siRNA clca1 and then were cultured for 72 hours. Cell lysates were collected for detection of ALP activity using the Alkaline Phosphatase Detection Kit or for ALP expression by western blot. The result shows that both ALP activity and expression were reduced significantly in CLCA1 knockdown cells. The histograms in A showed the relative intensity of CLCA1 38 KD, 90 KD, ALPI and β-catenin expressed as a ratio with respect to the GAPDH control. All results were from three independent experiments. ** p
Figure Legend Snippet: CLCA1 is required for spontaneous differentiation in Caco-2 cells. A. Caco-2 cells were transfected transiently with 0, 50, 100, 150 and 200 nM siRNA clca1 and blotted for CLCA1. siRNA clca1 at 100 nM or above effectively inhibited CLCA1 and downregulated expression of ALPI and β-catenin. B. Immunofluorescent staining showed the expression of β-catenin in confluent cultures of Caco-2 cells. β-catenin was located mainly in the nucleus of the cells at early stages of culture (2 days). After 10 days culture, β-catenin had translocated to the cell membrane. Knockdown of CLCA1 reduced distribution of β-catenin on the membrane. C. Caco-2 cells were treated with either siRNA negative control or siRNA clca1 and then were cultured for 72 hours. Cell lysates were collected for detection of ALP activity using the Alkaline Phosphatase Detection Kit or for ALP expression by western blot. The result shows that both ALP activity and expression were reduced significantly in CLCA1 knockdown cells. The histograms in A showed the relative intensity of CLCA1 38 KD, 90 KD, ALPI and β-catenin expressed as a ratio with respect to the GAPDH control. All results were from three independent experiments. ** p

Techniques Used: Transfection, Expressing, Staining, Negative Control, Cell Culture, ALP Assay, Activity Assay, Western Blot

40) Product Images from "Zika virus as an oncolytic treatment of human neuroblastoma cells requires CD24"

Article Title: Zika virus as an oncolytic treatment of human neuroblastoma cells requires CD24

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200358

Analysis of the role of CD24 in Zika-virus infected neuroblastoma cells. A) Western blot analysis of siRNA-mediated knock-down of CD24 expression in IMR-32 cells. Samples include Negative Control siRNA and CD24 siRNA. B) Western blot analysis of the expression of Envelope protein and NS1 (Non-Structural 1) protein in IMR-32 cells after siRNA-mediated knock-down of CD24 expression, 96 hours after Zika infection (MOI = 10). Samples include control cells treated with non-infected conditioned media and infected IMR-32 cells transfected with either Negative Control siRNA or CD24 siRNA. C) Western blot analysis of CD24 expression in the human neuroblastoma cell line SK-N-AS, comparing wild type (WT) to stably selected “Vector Only” (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). D) Western blot analysis of Zika NS1 protein expression 96 hours after Zika infection in CD24-stably expressing SK-N-AS cells, comparing wild type (WT) to stably selected Vector Only (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). GAPDH was used as a load control for all experiments. All results are representative of the combined data of experiments performed in triplicate.
Figure Legend Snippet: Analysis of the role of CD24 in Zika-virus infected neuroblastoma cells. A) Western blot analysis of siRNA-mediated knock-down of CD24 expression in IMR-32 cells. Samples include Negative Control siRNA and CD24 siRNA. B) Western blot analysis of the expression of Envelope protein and NS1 (Non-Structural 1) protein in IMR-32 cells after siRNA-mediated knock-down of CD24 expression, 96 hours after Zika infection (MOI = 10). Samples include control cells treated with non-infected conditioned media and infected IMR-32 cells transfected with either Negative Control siRNA or CD24 siRNA. C) Western blot analysis of CD24 expression in the human neuroblastoma cell line SK-N-AS, comparing wild type (WT) to stably selected “Vector Only” (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). D) Western blot analysis of Zika NS1 protein expression 96 hours after Zika infection in CD24-stably expressing SK-N-AS cells, comparing wild type (WT) to stably selected Vector Only (VO), CD24 variant 1 (V1), and CD24 variant 7 (V7). GAPDH was used as a load control for all experiments. All results are representative of the combined data of experiments performed in triplicate.

Techniques Used: Infection, Western Blot, Expressing, Negative Control, Transfection, Stable Transfection, Variant Assay, Plasmid Preparation

Analysis of CD24 expression in human neuroblastoma cells. A) Schematic of the alignment of CD24 splice variants 1 and 7. B C) Absolute quantification of CD24 expression by quantitative real-time PCR of total RNA acquired from neuroblastoma cells, measuring CD24 splice variants 1 (B) and 7 (C). Copy number values were normalized to the corresponding GAPDH values to determine the relative copy number. ** p > 0.05, Student’s t-test. D) Western blot analysis of CD24 expression in the total cell lysates of neuroblastoma cells. GAPDH was used as a loading control. All results are representative of the combined data of experiments performed in triplicate, with error bars representing standard deviation.
Figure Legend Snippet: Analysis of CD24 expression in human neuroblastoma cells. A) Schematic of the alignment of CD24 splice variants 1 and 7. B C) Absolute quantification of CD24 expression by quantitative real-time PCR of total RNA acquired from neuroblastoma cells, measuring CD24 splice variants 1 (B) and 7 (C). Copy number values were normalized to the corresponding GAPDH values to determine the relative copy number. ** p > 0.05, Student’s t-test. D) Western blot analysis of CD24 expression in the total cell lysates of neuroblastoma cells. GAPDH was used as a loading control. All results are representative of the combined data of experiments performed in triplicate, with error bars representing standard deviation.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

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  • 93
    Santa Cruz Biotechnology gapdh antibodies
    Effects of Meth on dopamine receptors and <t>sigma-1</t> receptor in CD4 + T-cells. ( A ) CD4 + T-cells were untreated or treated with 100 µM Meth for different time points (5 mins-24 hours), lysed and the protein extracts were analyzed for the expression of various dopamine receptors and sigma-1 receptor. <t>GAPDH</t> used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( B ) Fold change in the pixel density of sigma-1 receptor expression in ( A ). All values normalized to untreated sample. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( C ) CD4 + T-cells were untreated or treated with 10 µM sigma-1 receptor inhibitor (σ1 R inh.) for 1 hour, then treated with or without 100 µM Meth for 1 hour, followed by lysis and analysis of protein extracts for the indicated activated signaling molecules by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( D ) HIV-1 p24 titer on day 3 after HIV-1 infection in unstimulated and stimulated CD4 + T-cells pretreated with or without sigma-1 receptor inhibitor (σ1 R inh.) and treated in the presence or absence of Meth. Data represent the mean ± SD of 3 independent experiments (**p ≤ 0.01, ***p ≤ 0.001).
    Gapdh Antibodies, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2078 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology antibodies against glyceraldehyde 3 phosphate dehydrogenase gapdh
    Low concentration of Sal highly activates Akt. Hs578T cell extracts were collected at ( A ) 12 h and ( B ) 24 h after treatment with 0.5 μM Sal or from Dimethylsulfoxide (DMSO)-treated samples (Con). Western blot analyses were performed using antibodies against pJnk1, pAkt, Akt, PI3K, Jnk1, pp38, p38, pJak2, Jak2, pErk1/2, Erk1/2, pIKKα/β, Jak1, and glyceraldehyde 3-phosphate dehydrogenase <t>(GAPDH).</t>
    Antibodies Against Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology mouse monoclonal anti gapdh
    BID processing, mitochondrial depolarization and AIF nuclear traslocation in PES-treated PEL cells. ( a ) BID and LAMP2 expression in BC3 and BCBL1 untreated or treated with PES was analyzed by western blot. <t>β</t> -Actin was used as loading control. ( b ) MOMP assay was performed by using 40 nM TMRE for 15 min at 37 °C on PEL cells untreated or treated with PES and mean±S.D. is reported. ( c ) Western blot analysis for AIF, Lamin B and <t>GAPDH</t> was performed on cytosolic (C) and nuclear (N) fractions of control PEL cells or after treatment with PES. For all these experiments PES was used at 20 μ M for 24 h
    Mouse Monoclonal Anti Gapdh, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of Meth on dopamine receptors and sigma-1 receptor in CD4 + T-cells. ( A ) CD4 + T-cells were untreated or treated with 100 µM Meth for different time points (5 mins-24 hours), lysed and the protein extracts were analyzed for the expression of various dopamine receptors and sigma-1 receptor. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( B ) Fold change in the pixel density of sigma-1 receptor expression in ( A ). All values normalized to untreated sample. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( C ) CD4 + T-cells were untreated or treated with 10 µM sigma-1 receptor inhibitor (σ1 R inh.) for 1 hour, then treated with or without 100 µM Meth for 1 hour, followed by lysis and analysis of protein extracts for the indicated activated signaling molecules by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( D ) HIV-1 p24 titer on day 3 after HIV-1 infection in unstimulated and stimulated CD4 + T-cells pretreated with or without sigma-1 receptor inhibitor (σ1 R inh.) and treated in the presence or absence of Meth. Data represent the mean ± SD of 3 independent experiments (**p ≤ 0.01, ***p ≤ 0.001).

    Journal: Scientific Reports

    Article Title: Methamphetamine functions as a novel CD4+ T-cell activator via the sigma-1 receptor to enhance HIV-1 infection

    doi: 10.1038/s41598-018-35757-x

    Figure Lengend Snippet: Effects of Meth on dopamine receptors and sigma-1 receptor in CD4 + T-cells. ( A ) CD4 + T-cells were untreated or treated with 100 µM Meth for different time points (5 mins-24 hours), lysed and the protein extracts were analyzed for the expression of various dopamine receptors and sigma-1 receptor. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( B ) Fold change in the pixel density of sigma-1 receptor expression in ( A ). All values normalized to untreated sample. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). ( C ) CD4 + T-cells were untreated or treated with 10 µM sigma-1 receptor inhibitor (σ1 R inh.) for 1 hour, then treated with or without 100 µM Meth for 1 hour, followed by lysis and analysis of protein extracts for the indicated activated signaling molecules by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S3 . ( D ) HIV-1 p24 titer on day 3 after HIV-1 infection in unstimulated and stimulated CD4 + T-cells pretreated with or without sigma-1 receptor inhibitor (σ1 R inh.) and treated in the presence or absence of Meth. Data represent the mean ± SD of 3 independent experiments (**p ≤ 0.01, ***p ≤ 0.001).

    Article Snippet: GW182, D1DR, D2DR, D3DR, D4DR, Sigma-1 Receptor, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Expressing, Lysis, Western Blot, Infection

    Meth induced degradation of Ago1 and altered structural integrity of P-bodies: ( A ) CD4 + T-cells were untreated or treated with Meth (100 µM) for 0, 4 and 24 hours, lysed and Ago1 expression was analyzed by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S4 ( B ) CD4 + T-cell lysates in ( A ) were immunoprecipitated with Ago1 antibody and subjected to Western blot analysis using Ubiquitin antibody. Ago1 served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. ( C ) CD4 + T-cell lysates in (A) were immunoprecipitated with GW182 antibody (upper panel) or Ago1 antibody (lower panel) and subjected to Western blot analysis using Ago1 (upper panel) or GW182 (lower panel) antibodies. B-Actin served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S4 ( D ) Confocal images of GW182 and Ago1 interaction in CD4 + T-cells, untreated or treated with Meth (100 µM) for 24 hours. Scale bar = 10 µm. Results are representative of 3 independent experiments.

    Journal: Scientific Reports

    Article Title: Methamphetamine functions as a novel CD4+ T-cell activator via the sigma-1 receptor to enhance HIV-1 infection

    doi: 10.1038/s41598-018-35757-x

    Figure Lengend Snippet: Meth induced degradation of Ago1 and altered structural integrity of P-bodies: ( A ) CD4 + T-cells were untreated or treated with Meth (100 µM) for 0, 4 and 24 hours, lysed and Ago1 expression was analyzed by Western blotting. GAPDH used as a loading control. Full-length blots are presented in Supplementary Fig. S4 ( B ) CD4 + T-cell lysates in ( A ) were immunoprecipitated with Ago1 antibody and subjected to Western blot analysis using Ubiquitin antibody. Ago1 served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. ( C ) CD4 + T-cell lysates in (A) were immunoprecipitated with GW182 antibody (upper panel) or Ago1 antibody (lower panel) and subjected to Western blot analysis using Ago1 (upper panel) or GW182 (lower panel) antibodies. B-Actin served as a loading control; AbC = Antibody control, TCL = Total cell lysate. Results are representative of 3 independent experiments. Full-length blots are presented in Supplementary Fig. S4 ( D ) Confocal images of GW182 and Ago1 interaction in CD4 + T-cells, untreated or treated with Meth (100 µM) for 24 hours. Scale bar = 10 µm. Results are representative of 3 independent experiments.

    Article Snippet: GW182, D1DR, D2DR, D3DR, D4DR, Sigma-1 Receptor, and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

    Techniques: Expressing, Western Blot, Immunoprecipitation

    Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Journal: The EMBO Journal

    Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy

    doi: 10.15252/embj.201899576

    Figure Lengend Snippet: Unfolded Protein Response activation in TA UPR activation was analyzed by immunoblotting with indicated antibodies in TA muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, and p‐Eif2a protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Article Snippet: Reagents The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874).

    Techniques: Activation Assay, Mouse Assay

    Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation UPR activation was analyzed by immunoblotting with indicated antibodies in GC muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p‐Eif2a, and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Journal: The EMBO Journal

    Article Title: Lipin1 deficiency causes sarcoplasmic reticulum stress and chaperone‐responsive myopathy

    doi: 10.15252/embj.201899576

    Figure Lengend Snippet: Lipin1 deficiency triggers an ER stress in muscles and leads to Unfolded Protein Response activation UPR activation was analyzed by immunoblotting with indicated antibodies in GC muscles of 4‐month‐old HSA‐Cre ( n = 7) and control ( n = 4) mice. Quantification by densitometric analyses of Fgf21, Bip, sXbp1 Atf6 cleaved form, p‐Eif2a, and Chop protein levels is presented as a graph. Data are normalized to Gapdh and expressed as a fold change relative to the control mice. Data are mean ± SEM (* P

    Article Snippet: Reagents The following primary antibodies were used: anti‐p62 (SQSTM) (Abnova), anti‐LAMP2 (Abcam), anti‐FGF21 (abcam), anti‐Bip (BD Biosciences), anti‐Gapdh (Santa Cruz), anti‐SREBP1c (Santa Cruz), anti‐SREBP2 (abcam), anti‐ATF6 (abcam), anti‐LC3 (Nanotools), anti‐Tom20 (SantaCruz), anti‐lipin1 (SantaCruz, sc‐376874).

    Techniques: Activation Assay, Mouse Assay

    Low concentration of Sal highly activates Akt. Hs578T cell extracts were collected at ( A ) 12 h and ( B ) 24 h after treatment with 0.5 μM Sal or from Dimethylsulfoxide (DMSO)-treated samples (Con). Western blot analyses were performed using antibodies against pJnk1, pAkt, Akt, PI3K, Jnk1, pp38, p38, pJak2, Jak2, pErk1/2, Erk1/2, pIKKα/β, Jak1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

    Journal: International Journal of Molecular Sciences

    Article Title: Low Amount of Salinomycin Greatly Increases Akt Activation, but Reduces Activated p70S6K Levels

    doi: 10.3390/ijms140917304

    Figure Lengend Snippet: Low concentration of Sal highly activates Akt. Hs578T cell extracts were collected at ( A ) 12 h and ( B ) 24 h after treatment with 0.5 μM Sal or from Dimethylsulfoxide (DMSO)-treated samples (Con). Western blot analyses were performed using antibodies against pJnk1, pAkt, Akt, PI3K, Jnk1, pp38, p38, pJak2, Jak2, pErk1/2, Erk1/2, pIKKα/β, Jak1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

    Article Snippet: Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphorylated p38, p38, Erk1/2, Jak1, Jak2, survivin, and pRb were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Concentration Assay, Western Blot

    BID processing, mitochondrial depolarization and AIF nuclear traslocation in PES-treated PEL cells. ( a ) BID and LAMP2 expression in BC3 and BCBL1 untreated or treated with PES was analyzed by western blot. β -Actin was used as loading control. ( b ) MOMP assay was performed by using 40 nM TMRE for 15 min at 37 °C on PEL cells untreated or treated with PES and mean±S.D. is reported. ( c ) Western blot analysis for AIF, Lamin B and GAPDH was performed on cytosolic (C) and nuclear (N) fractions of control PEL cells or after treatment with PES. For all these experiments PES was used at 20 μ M for 24 h

    Journal: Cell Death & Disease

    Article Title: HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma

    doi: 10.1038/cddis.2013.263

    Figure Lengend Snippet: BID processing, mitochondrial depolarization and AIF nuclear traslocation in PES-treated PEL cells. ( a ) BID and LAMP2 expression in BC3 and BCBL1 untreated or treated with PES was analyzed by western blot. β -Actin was used as loading control. ( b ) MOMP assay was performed by using 40 nM TMRE for 15 min at 37 °C on PEL cells untreated or treated with PES and mean±S.D. is reported. ( c ) Western blot analysis for AIF, Lamin B and GAPDH was performed on cytosolic (C) and nuclear (N) fractions of control PEL cells or after treatment with PES. For all these experiments PES was used at 20 μ M for 24 h

    Article Snippet: Antibodies In this work, the following antibodies were used: rabbit polyclonal anti-LC3 (Novus Biologicals, Cambridge, UK; cat. no. NB100-2220SS), mouse monoclonal anti-p62 (BD Transduction Laboratories, San Jose, CA, USA; cat. no. 610833), goat polyclonal anti-lamin B (Santa Cruz Biotechnology, Europe; cat. no. sc-6216), mouse monoclonal anti-caspase8 (Cell Signaling, Boston, MA, USA; cat. no. 4927), goat polyclonal anti-caspase 3 (Santa Cruz Biotechnology; cat. no. sc-1225), mouse monoclonal anti-HSP70 (Santa Cruz Biotechnology; cat. no. sc-66049), rabbit polyclonal anti-BID (Cell Signaling; cat. no. 2002), rabbit polyclonal anti-AIF (Santa Cruz Biotechnology; cat. no. sc-5586), goat polyclonal anti-cathepsin D (Santa Cruz Biotechnology; cat. no. sc-6487), mouse monoclonal anti-CD86 (BD Pharmingen; cat. no. 558703), mouse monoclonal anti-β -actin (Sigma; cat. no A2228) and mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology; cat. no. sc-137179).

    Techniques: Expressing, Western Blot