Structured Review

Abcam rab7
Vesicles associated with the nuclear envelope are derived from early endosomes. ( a ) Cells pre-treated with Brefeldin A (BFA) or dimethylsulfoxide (DMSO) both before and during incubation with Alexa 488-PE were stained for giantin (scale bar, 5 μm). ( b ) Percentage of cells displaying NAE structures. Representative of three independent experiments (error bars indicate s.d.). ( c ) Average number of NAE structures by nuclei. Representative of three independent experiments (error bars indicate s.d.). ( d ) Co-labelling of PE with early endosomal marker EEA1. ( e ) Co-labelling with the late endosomal marker <t>Rab7</t> (scale bar, 10 μm, × 5 image: scale bar, 2 μm). Data shown in c and d are from three experiments.
Rab7, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 1651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Nuclear envelope-associated endosomes deliver surface proteins to the nucleus"

Article Title: Nuclear envelope-associated endosomes deliver surface proteins to the nucleus

Journal: Nature Communications

doi: 10.1038/ncomms9218

Vesicles associated with the nuclear envelope are derived from early endosomes. ( a ) Cells pre-treated with Brefeldin A (BFA) or dimethylsulfoxide (DMSO) both before and during incubation with Alexa 488-PE were stained for giantin (scale bar, 5 μm). ( b ) Percentage of cells displaying NAE structures. Representative of three independent experiments (error bars indicate s.d.). ( c ) Average number of NAE structures by nuclei. Representative of three independent experiments (error bars indicate s.d.). ( d ) Co-labelling of PE with early endosomal marker EEA1. ( e ) Co-labelling with the late endosomal marker Rab7 (scale bar, 10 μm, × 5 image: scale bar, 2 μm). Data shown in c and d are from three experiments.
Figure Legend Snippet: Vesicles associated with the nuclear envelope are derived from early endosomes. ( a ) Cells pre-treated with Brefeldin A (BFA) or dimethylsulfoxide (DMSO) both before and during incubation with Alexa 488-PE were stained for giantin (scale bar, 5 μm). ( b ) Percentage of cells displaying NAE structures. Representative of three independent experiments (error bars indicate s.d.). ( c ) Average number of NAE structures by nuclei. Representative of three independent experiments (error bars indicate s.d.). ( d ) Co-labelling of PE with early endosomal marker EEA1. ( e ) Co-labelling with the late endosomal marker Rab7 (scale bar, 10 μm, × 5 image: scale bar, 2 μm). Data shown in c and d are from three experiments.

Techniques Used: Derivative Assay, Incubation, Staining, Marker

2) Product Images from "Nuclear envelope-associated endosomes deliver surface proteins to the nucleus"

Article Title: Nuclear envelope-associated endosomes deliver surface proteins to the nucleus

Journal: Nature Communications

doi: 10.1038/ncomms9218

PE requires SUN1/2 and the translocon complex for transport to the nucleoplasm. ( a ) Flowchart and ( b ) results of siRNA screening; PE and Nu-T detection as in Fig. 4 . Representative of five different replicates. ( c – e ) MG63 cell fractionation after Sec61B or SUN2 knockdown. ( c ) Fractionation controls (as in Fig. 4 ). ( d ) PE subcellular distribution in MG63 cells after Sec61B and SUN2 knockdown. Representative of three independent repeats. ( e ) Quantification of PE in nuclear fraction after Sec61B and SUN2 knockdown. Error bars at s.d. t -Test type 2, two tails. ( f ) Co-labelling of PE with EGFR. Nucleus is delimited by yellow circle. Scale bar, 10 μm. ( g ) A431 cells fractionation. ( h ) Input loading control of A431 protein extract. ( i ) EGFR nuclear distribution in A431 cells after Sec61B and SUN2 knockdown. Representative of three independent repeats. ( j ) Quantification of EGFR in nuclear fraction. Error bars at s.d. t -Test type 2, two tails. ( k ) Two intracellular routes of cargos. After endocytosis, NAE dock on the nucleus, discharging their content into the nucleoplasm in a SUN1/2- and Sec61 translocon-dependent manner.
Figure Legend Snippet: PE requires SUN1/2 and the translocon complex for transport to the nucleoplasm. ( a ) Flowchart and ( b ) results of siRNA screening; PE and Nu-T detection as in Fig. 4 . Representative of five different replicates. ( c – e ) MG63 cell fractionation after Sec61B or SUN2 knockdown. ( c ) Fractionation controls (as in Fig. 4 ). ( d ) PE subcellular distribution in MG63 cells after Sec61B and SUN2 knockdown. Representative of three independent repeats. ( e ) Quantification of PE in nuclear fraction after Sec61B and SUN2 knockdown. Error bars at s.d. t -Test type 2, two tails. ( f ) Co-labelling of PE with EGFR. Nucleus is delimited by yellow circle. Scale bar, 10 μm. ( g ) A431 cells fractionation. ( h ) Input loading control of A431 protein extract. ( i ) EGFR nuclear distribution in A431 cells after Sec61B and SUN2 knockdown. Representative of three independent repeats. ( j ) Quantification of EGFR in nuclear fraction. Error bars at s.d. t -Test type 2, two tails. ( k ) Two intracellular routes of cargos. After endocytosis, NAE dock on the nucleus, discharging their content into the nucleoplasm in a SUN1/2- and Sec61 translocon-dependent manner.

Techniques Used: Cell Fractionation, Fractionation

3) Product Images from "Tetrandrine Suppresses Lipopolysaccharide-Induced Microglial Activation by Inhibiting NF-κB and ERK Signaling Pathways in BV2 Cells"

Article Title: Tetrandrine Suppresses Lipopolysaccharide-Induced Microglial Activation by Inhibiting NF-κB and ERK Signaling Pathways in BV2 Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0102522

Effect of TET on LPS-stimulated NF-κB activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Then, the cells were lysed for protein extraction.   Fig. 6a : p-p65, p65, p-IKK, IKK, and  β -actin expression were measured by Western blotting analysis.   Fig. 6b–6c : The ratio of densitometry values of total and phosphorylated protein was normalized to each respective control group.  β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. *  P
Figure Legend Snippet: Effect of TET on LPS-stimulated NF-κB activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Then, the cells were lysed for protein extraction. Fig. 6a : p-p65, p65, p-IKK, IKK, and β -actin expression were measured by Western blotting analysis. Fig. 6b–6c : The ratio of densitometry values of total and phosphorylated protein was normalized to each respective control group. β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. * P

Techniques Used: Activity Assay, Incubation, Protein Extraction, Expressing, Western Blot

Effect of TET on LPS-stimulated MAPK activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h.Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Thereafter, the cells were lysed for protein extraction.   Fig. 7a : Western blot assay was performed to evaluate the expression of total and phosphorylated forms of ERK1/2, JNK, and p38.  β -actin was applied as the internal control.   Fig. 7b : The ratio of densitometry values of total and phosphorylated forms of the protein was normalized to each respective control group.  β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. *  P
Figure Legend Snippet: Effect of TET on LPS-stimulated MAPK activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h.Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Thereafter, the cells were lysed for protein extraction. Fig. 7a : Western blot assay was performed to evaluate the expression of total and phosphorylated forms of ERK1/2, JNK, and p38. β -actin was applied as the internal control. Fig. 7b : The ratio of densitometry values of total and phosphorylated forms of the protein was normalized to each respective control group. β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. * P

Techniques Used: Activity Assay, Incubation, Protein Extraction, Western Blot, Expressing

Effect of TET on CD 11b expression in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h.The cells were lysed for protein extraction.   Fig. 4a : CD11b expression was measured by Western blotting analysis.   Fig. 4b : The ratio of densitometry values of CD11b and  β -actin was normalized to each respective control group. Results were the mean ± S.D. from three independent experiments performed in triplicate. * P
Figure Legend Snippet: Effect of TET on CD 11b expression in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h.The cells were lysed for protein extraction. Fig. 4a : CD11b expression was measured by Western blotting analysis. Fig. 4b : The ratio of densitometry values of CD11b and β -actin was normalized to each respective control group. Results were the mean ± S.D. from three independent experiments performed in triplicate. * P

Techniques Used: Expressing, Incubation, Protein Extraction, Western Blot

4) Product Images from "Exo70 is transcriptionally up-regulated by hepatic nuclear factor 4α and contributes to cell cycle control in hepatoma cells"

Article Title: Exo70 is transcriptionally up-regulated by hepatic nuclear factor 4α and contributes to cell cycle control in hepatoma cells

Journal: Oncotarget

doi: 10.18632/oncotarget.7133

HNF4α regulates the expression of Exo70 protein ( A – B ) Knocking down HNF4α decreased the protein and mRNA expression of Exo70. Hep G2 cells were transiently transfected with pLL3.7 vector or pLL3.7-shHNF4α plasmid. Cells were harvested 48 h after transfection and the whole cell lysate and the total RNA were prepared then subjected to Western blot (A) and real time PCR (B) analysis. ( C – D ) Ectopically expressed HNF4α upregulated the protein and mRNA expression level of Exo70 in cholangiocarcinoma cell line QBC939. Cells were transfected with pCMV10 vector or Flag-HNF4α plasmid, and harvested 48 h after transfection and subjected to Western blot (C) and real time PCR (D) analysis. Protein expression of Exo70, HNF4α and β-actin (used as loading control) were detected and quantified by densitometry; bar graph (A, C, down panel) showed the ratios of Exo70 or HNF4α to β-actin, and the basal level in the vector group was normalized to 1. GAPDH was used as normalization control for real time PCR. Data were presented as the means ± s.e.m. from three independent experiments. Differences between two groups as indicated in the graph were assessed by a two tailed unpaired Student's t -test, * p
Figure Legend Snippet: HNF4α regulates the expression of Exo70 protein ( A – B ) Knocking down HNF4α decreased the protein and mRNA expression of Exo70. Hep G2 cells were transiently transfected with pLL3.7 vector or pLL3.7-shHNF4α plasmid. Cells were harvested 48 h after transfection and the whole cell lysate and the total RNA were prepared then subjected to Western blot (A) and real time PCR (B) analysis. ( C – D ) Ectopically expressed HNF4α upregulated the protein and mRNA expression level of Exo70 in cholangiocarcinoma cell line QBC939. Cells were transfected with pCMV10 vector or Flag-HNF4α plasmid, and harvested 48 h after transfection and subjected to Western blot (C) and real time PCR (D) analysis. Protein expression of Exo70, HNF4α and β-actin (used as loading control) were detected and quantified by densitometry; bar graph (A, C, down panel) showed the ratios of Exo70 or HNF4α to β-actin, and the basal level in the vector group was normalized to 1. GAPDH was used as normalization control for real time PCR. Data were presented as the means ± s.e.m. from three independent experiments. Differences between two groups as indicated in the graph were assessed by a two tailed unpaired Student's t -test, * p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

5) Product Images from "AmotL2 integrates polarity and junctional cues to modulate cell shape"

Article Title: AmotL2 integrates polarity and junctional cues to modulate cell shape

Journal: Scientific Reports

doi: 10.1038/s41598-017-07968-1

AmotL2 localization to cell-cell junctions is Par3 dependent. ( a,b ) In MS-1 cells treated with control siRNA, both AmotL2 and Par3 (green) localize to β-catenin (red) positive cellular junctions. In Par3 siRNA treated cells, AmotL2 protein was absent from β-catenin positive junctions. In MS-1 cells treated with AmotL2 siRNA, Par3 junctional localization was unaffected. Insets show high magnification of cell-cell junctions. Scale bar 10 μm. ( c ) Quantification of AmotL2 co-localization to β-catenin in control and Par3 siRNA treated MS-1 cells. N(control siRNA) = 64 cells, N( Par3 siRNA) = 42 cells, p = 8.80 × 10 −5 . ( d ) Quantification of Par3 co-localization to β-catenin in control and AmotL2 siRNA treated MS-1 cells. N(control siRNA) = 38 cells, N( AmotL2 siRNA) = 70 cells, p = 0.87, Error bars indicate standard deviation (s.d.), N.S. = Non Significant, ***p ≤ 0.001. ( e ) co-IP demonstrating the association of both Par3 and AmotL2 to the E-cadherin protein complex. Interaction of AmotL2 to E-cadherin and MAGI-1/ β-actin was dependent on Par3.
Figure Legend Snippet: AmotL2 localization to cell-cell junctions is Par3 dependent. ( a,b ) In MS-1 cells treated with control siRNA, both AmotL2 and Par3 (green) localize to β-catenin (red) positive cellular junctions. In Par3 siRNA treated cells, AmotL2 protein was absent from β-catenin positive junctions. In MS-1 cells treated with AmotL2 siRNA, Par3 junctional localization was unaffected. Insets show high magnification of cell-cell junctions. Scale bar 10 μm. ( c ) Quantification of AmotL2 co-localization to β-catenin in control and Par3 siRNA treated MS-1 cells. N(control siRNA) = 64 cells, N( Par3 siRNA) = 42 cells, p = 8.80 × 10 −5 . ( d ) Quantification of Par3 co-localization to β-catenin in control and AmotL2 siRNA treated MS-1 cells. N(control siRNA) = 38 cells, N( AmotL2 siRNA) = 70 cells, p = 0.87, Error bars indicate standard deviation (s.d.), N.S. = Non Significant, ***p ≤ 0.001. ( e ) co-IP demonstrating the association of both Par3 and AmotL2 to the E-cadherin protein complex. Interaction of AmotL2 to E-cadherin and MAGI-1/ β-actin was dependent on Par3.

Techniques Used: Mass Spectrometry, Standard Deviation, Co-Immunoprecipitation Assay

Related Articles

Western Blot:

Article Title: Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons
Article Snippet: .. The following primary antibodies were used for western blot analysis: rabbit anti-Rab7 (ab137029, Abcam), rabbit anti-Rpl10A (16681-1-AP, Proteintech), mouse anti-Rps3A (ab194670, Abcam), mouse anti-FXR (gift from Dr. Khandjian), rabbit anti-SFPQ (ab38148, Abcam), rabbit anti-Vg1RBP (gift from Dr. Standart), rabbit anti-mTOR (#2983, Cell Signaling). .. Immunohistochemistry Retinal cultures were fixed in 2% formaldehyde/7.5% sucrose in PBS for 20 minutes at 20°C.

Article Title: Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing
Article Snippet: .. Antibodies Rabbit anti-Na+ /K+ ATPase (1:1000; 3010S; Cell Signaling Technology), mouse anti-EEA1(1:1000; ab70521; Abcam); rabbit anti-Rab7 (1;1000; ab137029; Abcam); rabbit anti-GAPDH (1:1000; 5174S; Cell Signaling Technology), rabbit anti-E . tarda OmpA polyclonal antibody (1:500; custom-made; Genscript Biotech, Piscataway, NJ, USA), rabbit anti-E . tarda EthA polyclonal antibody (1: 1000; custom-made; Genscript), and mouse anti-RNAP monoclonal antibody (1:5000; Santa Cruz Biotechnology) were used for western blotting or immunofluorescence. .. Supporting information Hemolysin-overexpressing E. tarda strains promoted caspase-4 inflammasome activation, related to . ( A ) LDH release detected in wild-type HeLa cells infected with the gene-defined mutant library of E . tarda at MOI = 100 for the indicated time periods, only partial data were showed here. ( B ) Cell morphology with PI staining of HT-29 cells infected with indicated E . tarda strains (MOI = 25, 4 hpi), scale = 20 μm. ( C - D ) LDH release (C) and IL-18 secretion (D) detected in wild-type and Caspase-4 -/- HT-29 cells infected with indicated E . tarda strains (MOI = 25, 4 hpi).

Article Title: Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing
Article Snippet: .. Rabbit anti-Na+ /K+ ATPase (1:1000; 3010S; Cell Signaling Technology), mouse anti-EEA1(1:1000; ab70521; Abcam); rabbit anti-Rab7 (1;1000; ab137029; Abcam); rabbit anti-GAPDH (1:1000; 5174S; Cell Signaling Technology), rabbit anti- E . tarda OmpA polyclonal antibody (1:500; custom-made; Genscript Biotech, Piscataway, NJ, USA), rabbit anti- E . tarda EthA polyclonal antibody (1: 1000; custom-made; Genscript), and mouse anti-RNAP monoclonal antibody (1:5000; Santa Cruz Biotechnology) were used for western blotting or immunofluorescence. .. Hemolysin-overexpressing E. tarda strains promoted caspase-4 inflammasome activation, related to . ( A ) LDH release detected in wild-type HeLa cells infected with the gene-defined mutant library of E . tarda at MOI = 100 for the indicated time periods, only partial data were showed here. ( B ) Cell morphology with PI staining of HT-29 cells infected with indicated E . tarda strains (MOI = 25, 4 hpi), scale = 20 μm. ( C - D ) LDH release (C) and IL-18 secretion (D) detected in wild-type and Caspase-4 -/- HT-29 cells infected with indicated E . tarda strains (MOI = 25, 4 hpi).

Incubation:

Article Title: Nuclear envelope-associated endosomes deliver surface proteins to the nucleus
Article Snippet: .. Membranes were incubated with actin (#3280, 1:5,000, Abcam), β-tubulin (#ab6046, 1:5,000 Abcam), EEA1 (#E7659, 1:1,000, Sigma-Aldrich), EGFR (#2232L, 1:1,000, Cell Signaling Technology), histone H1 (#sc-10806, 1:1,000, Santa Cruz), PDI (#RL77, 1:15,000, Abcam), LRP1 (#L2420, 1:1,000, Sigma-Aldrich), Rab7 (#ab137029, 1:1,000, Abcam), Sec61B (#ab78276, 1:5,000, Abcam), streptavidin-horseradish peroxidase (HRP; #21130, 1:10,000, Thermo Fisher Scientific) and SUN2 (# HPA001209, 1:2,000, Sigma-Aldrich). ..

other:

Article Title: Cathepsin S attenuates endosomal EGFR signalling: A mechanical rationale for the combination of cathepsin S and EGFR tyrosine kinase inhibitors
Article Snippet: An antibody against Rab7 (#ab137029) was purchased from Abcam.

Immunofluorescence:

Article Title: Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing
Article Snippet: .. Antibodies Rabbit anti-Na+ /K+ ATPase (1:1000; 3010S; Cell Signaling Technology), mouse anti-EEA1(1:1000; ab70521; Abcam); rabbit anti-Rab7 (1;1000; ab137029; Abcam); rabbit anti-GAPDH (1:1000; 5174S; Cell Signaling Technology), rabbit anti-E . tarda OmpA polyclonal antibody (1:500; custom-made; Genscript Biotech, Piscataway, NJ, USA), rabbit anti-E . tarda EthA polyclonal antibody (1: 1000; custom-made; Genscript), and mouse anti-RNAP monoclonal antibody (1:5000; Santa Cruz Biotechnology) were used for western blotting or immunofluorescence. .. Supporting information Hemolysin-overexpressing E. tarda strains promoted caspase-4 inflammasome activation, related to . ( A ) LDH release detected in wild-type HeLa cells infected with the gene-defined mutant library of E . tarda at MOI = 100 for the indicated time periods, only partial data were showed here. ( B ) Cell morphology with PI staining of HT-29 cells infected with indicated E . tarda strains (MOI = 25, 4 hpi), scale = 20 μm. ( C - D ) LDH release (C) and IL-18 secretion (D) detected in wild-type and Caspase-4 -/- HT-29 cells infected with indicated E . tarda strains (MOI = 25, 4 hpi).

Article Title: Dysregulated hemolysin liberates bacterial outer membrane vesicles for cytosolic lipopolysaccharide sensing
Article Snippet: .. Rabbit anti-Na+ /K+ ATPase (1:1000; 3010S; Cell Signaling Technology), mouse anti-EEA1(1:1000; ab70521; Abcam); rabbit anti-Rab7 (1;1000; ab137029; Abcam); rabbit anti-GAPDH (1:1000; 5174S; Cell Signaling Technology), rabbit anti- E . tarda OmpA polyclonal antibody (1:500; custom-made; Genscript Biotech, Piscataway, NJ, USA), rabbit anti- E . tarda EthA polyclonal antibody (1: 1000; custom-made; Genscript), and mouse anti-RNAP monoclonal antibody (1:5000; Santa Cruz Biotechnology) were used for western blotting or immunofluorescence. .. Hemolysin-overexpressing E. tarda strains promoted caspase-4 inflammasome activation, related to . ( A ) LDH release detected in wild-type HeLa cells infected with the gene-defined mutant library of E . tarda at MOI = 100 for the indicated time periods, only partial data were showed here. ( B ) Cell morphology with PI staining of HT-29 cells infected with indicated E . tarda strains (MOI = 25, 4 hpi), scale = 20 μm. ( C - D ) LDH release (C) and IL-18 secretion (D) detected in wild-type and Caspase-4 -/- HT-29 cells infected with indicated E . tarda strains (MOI = 25, 4 hpi).

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    Abcam β actin
    Effect of TET on LPS-stimulated NF-κB activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Then, the cells were lysed for protein extraction.   Fig. 6a : p-p65, p65, p-IKK, IKK, and  β -actin expression were measured by Western blotting analysis.   Fig. 6b–6c : The ratio of densitometry values of total and phosphorylated protein was normalized to each respective control group.  β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. *  P
    β Actin, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1351 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of TET on LPS-stimulated NF-κB activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Then, the cells were lysed for protein extraction.   Fig. 6a : p-p65, p65, p-IKK, IKK, and  β -actin expression were measured by Western blotting analysis.   Fig. 6b–6c : The ratio of densitometry values of total and phosphorylated protein was normalized to each respective control group.  β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. *  P

    Journal: PLoS ONE

    Article Title: Tetrandrine Suppresses Lipopolysaccharide-Induced Microglial Activation by Inhibiting NF-κB and ERK Signaling Pathways in BV2 Cells

    doi: 10.1371/journal.pone.0102522

    Figure Lengend Snippet: Effect of TET on LPS-stimulated NF-κB activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Then, the cells were lysed for protein extraction. Fig. 6a : p-p65, p65, p-IKK, IKK, and β -actin expression were measured by Western blotting analysis. Fig. 6b–6c : The ratio of densitometry values of total and phosphorylated protein was normalized to each respective control group. β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. * P

    Article Snippet: The membranes were blocked by StartingBlock T20 (Pierce Biotechnology) prior to incubation in primary antibodies: CD11b (1∶300, #ab8878, AbCam), p-P65 (1∶1000, #13346, Cell Signaling), P65 (1∶800, #ab7970, AbCam), p-IKK (1∶1000, #2697, Cell Signaling), IKK(1∶250, #ab54626, AbCam), p-ERK (1∶2000, #4370, Cell Signaling), ERK(1∶1000, #4696,Cell Signaling), p-JNK (1∶1000, #9251, Cell Signaling), JNK(1∶1000, #9252, Cell Signaling), p-P38 (1∶1000, #4511, Cell Signaling), and P38(1∶500, #9212, Cell Signaling) and β-actin (1∶400, #ab3280, AbCam) overnight at 4°C.

    Techniques: Activity Assay, Incubation, Protein Extraction, Expressing, Western Blot

    Effect of TET on LPS-stimulated MAPK activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h.Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Thereafter, the cells were lysed for protein extraction.   Fig. 7a : Western blot assay was performed to evaluate the expression of total and phosphorylated forms of ERK1/2, JNK, and p38.  β -actin was applied as the internal control.   Fig. 7b : The ratio of densitometry values of total and phosphorylated forms of the protein was normalized to each respective control group.  β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. *  P

    Journal: PLoS ONE

    Article Title: Tetrandrine Suppresses Lipopolysaccharide-Induced Microglial Activation by Inhibiting NF-κB and ERK Signaling Pathways in BV2 Cells

    doi: 10.1371/journal.pone.0102522

    Figure Lengend Snippet: Effect of TET on LPS-stimulated MAPK activity in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h.Then, LPS was added to each plate and incubated at 37°C for an additional 24 h. Thereafter, the cells were lysed for protein extraction. Fig. 7a : Western blot assay was performed to evaluate the expression of total and phosphorylated forms of ERK1/2, JNK, and p38. β -actin was applied as the internal control. Fig. 7b : The ratio of densitometry values of total and phosphorylated forms of the protein was normalized to each respective control group. β -actin was applied as the internal control. Results were the mean ± S.D. from three independent experiments performed in triplicate. * P

    Article Snippet: The membranes were blocked by StartingBlock T20 (Pierce Biotechnology) prior to incubation in primary antibodies: CD11b (1∶300, #ab8878, AbCam), p-P65 (1∶1000, #13346, Cell Signaling), P65 (1∶800, #ab7970, AbCam), p-IKK (1∶1000, #2697, Cell Signaling), IKK(1∶250, #ab54626, AbCam), p-ERK (1∶2000, #4370, Cell Signaling), ERK(1∶1000, #4696,Cell Signaling), p-JNK (1∶1000, #9251, Cell Signaling), JNK(1∶1000, #9252, Cell Signaling), p-P38 (1∶1000, #4511, Cell Signaling), and P38(1∶500, #9212, Cell Signaling) and β-actin (1∶400, #ab3280, AbCam) overnight at 4°C.

    Techniques: Activity Assay, Incubation, Protein Extraction, Western Blot, Expressing

    Effect of TET on CD 11b expression in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h.The cells were lysed for protein extraction.   Fig. 4a : CD11b expression was measured by Western blotting analysis.   Fig. 4b : The ratio of densitometry values of CD11b and  β -actin was normalized to each respective control group. Results were the mean ± S.D. from three independent experiments performed in triplicate. * P

    Journal: PLoS ONE

    Article Title: Tetrandrine Suppresses Lipopolysaccharide-Induced Microglial Activation by Inhibiting NF-κB and ERK Signaling Pathways in BV2 Cells

    doi: 10.1371/journal.pone.0102522

    Figure Lengend Snippet: Effect of TET on CD 11b expression in BV2 cells. BV2 cells were treated with TET at working concentrations for 2 h. Then, LPS was added to each plate and incubated at 37°C for an additional 24 h.The cells were lysed for protein extraction. Fig. 4a : CD11b expression was measured by Western blotting analysis. Fig. 4b : The ratio of densitometry values of CD11b and β -actin was normalized to each respective control group. Results were the mean ± S.D. from three independent experiments performed in triplicate. * P

    Article Snippet: The membranes were blocked by StartingBlock T20 (Pierce Biotechnology) prior to incubation in primary antibodies: CD11b (1∶300, #ab8878, AbCam), p-P65 (1∶1000, #13346, Cell Signaling), P65 (1∶800, #ab7970, AbCam), p-IKK (1∶1000, #2697, Cell Signaling), IKK(1∶250, #ab54626, AbCam), p-ERK (1∶2000, #4370, Cell Signaling), ERK(1∶1000, #4696,Cell Signaling), p-JNK (1∶1000, #9251, Cell Signaling), JNK(1∶1000, #9252, Cell Signaling), p-P38 (1∶1000, #4511, Cell Signaling), and P38(1∶500, #9212, Cell Signaling) and β-actin (1∶400, #ab3280, AbCam) overnight at 4°C.

    Techniques: Expressing, Incubation, Protein Extraction, Western Blot

    HNF4α regulates the expression of Exo70 protein ( A – B ) Knocking down HNF4α decreased the protein and mRNA expression of Exo70. Hep G2 cells were transiently transfected with pLL3.7 vector or pLL3.7-shHNF4α plasmid. Cells were harvested 48 h after transfection and the whole cell lysate and the total RNA were prepared then subjected to Western blot (A) and real time PCR (B) analysis. ( C – D ) Ectopically expressed HNF4α upregulated the protein and mRNA expression level of Exo70 in cholangiocarcinoma cell line QBC939. Cells were transfected with pCMV10 vector or Flag-HNF4α plasmid, and harvested 48 h after transfection and subjected to Western blot (C) and real time PCR (D) analysis. Protein expression of Exo70, HNF4α and β-actin (used as loading control) were detected and quantified by densitometry; bar graph (A, C, down panel) showed the ratios of Exo70 or HNF4α to β-actin, and the basal level in the vector group was normalized to 1. GAPDH was used as normalization control for real time PCR. Data were presented as the means ± s.e.m. from three independent experiments. Differences between two groups as indicated in the graph were assessed by a two tailed unpaired Student's t -test, * p

    Journal: Oncotarget

    Article Title: Exo70 is transcriptionally up-regulated by hepatic nuclear factor 4α and contributes to cell cycle control in hepatoma cells

    doi: 10.18632/oncotarget.7133

    Figure Lengend Snippet: HNF4α regulates the expression of Exo70 protein ( A – B ) Knocking down HNF4α decreased the protein and mRNA expression of Exo70. Hep G2 cells were transiently transfected with pLL3.7 vector or pLL3.7-shHNF4α plasmid. Cells were harvested 48 h after transfection and the whole cell lysate and the total RNA were prepared then subjected to Western blot (A) and real time PCR (B) analysis. ( C – D ) Ectopically expressed HNF4α upregulated the protein and mRNA expression level of Exo70 in cholangiocarcinoma cell line QBC939. Cells were transfected with pCMV10 vector or Flag-HNF4α plasmid, and harvested 48 h after transfection and subjected to Western blot (C) and real time PCR (D) analysis. Protein expression of Exo70, HNF4α and β-actin (used as loading control) were detected and quantified by densitometry; bar graph (A, C, down panel) showed the ratios of Exo70 or HNF4α to β-actin, and the basal level in the vector group was normalized to 1. GAPDH was used as normalization control for real time PCR. Data were presented as the means ± s.e.m. from three independent experiments. Differences between two groups as indicated in the graph were assessed by a two tailed unpaired Student's t -test, * p

    Article Snippet: Mouse anti-β-actin antibody (cat. #ab3280) was purchased from Abcam.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Two Tailed Test

    AmotL2 localization to cell-cell junctions is Par3 dependent. ( a,b ) In MS-1 cells treated with control siRNA, both AmotL2 and Par3 (green) localize to β-catenin (red) positive cellular junctions. In Par3 siRNA treated cells, AmotL2 protein was absent from β-catenin positive junctions. In MS-1 cells treated with AmotL2 siRNA, Par3 junctional localization was unaffected. Insets show high magnification of cell-cell junctions. Scale bar 10 μm. ( c ) Quantification of AmotL2 co-localization to β-catenin in control and Par3 siRNA treated MS-1 cells. N(control siRNA) = 64 cells, N( Par3 siRNA) = 42 cells, p = 8.80 × 10 −5 . ( d ) Quantification of Par3 co-localization to β-catenin in control and AmotL2 siRNA treated MS-1 cells. N(control siRNA) = 38 cells, N( AmotL2 siRNA) = 70 cells, p = 0.87, Error bars indicate standard deviation (s.d.), N.S. = Non Significant, ***p ≤ 0.001. ( e ) co-IP demonstrating the association of both Par3 and AmotL2 to the E-cadherin protein complex. Interaction of AmotL2 to E-cadherin and MAGI-1/ β-actin was dependent on Par3.

    Journal: Scientific Reports

    Article Title: AmotL2 integrates polarity and junctional cues to modulate cell shape

    doi: 10.1038/s41598-017-07968-1

    Figure Lengend Snippet: AmotL2 localization to cell-cell junctions is Par3 dependent. ( a,b ) In MS-1 cells treated with control siRNA, both AmotL2 and Par3 (green) localize to β-catenin (red) positive cellular junctions. In Par3 siRNA treated cells, AmotL2 protein was absent from β-catenin positive junctions. In MS-1 cells treated with AmotL2 siRNA, Par3 junctional localization was unaffected. Insets show high magnification of cell-cell junctions. Scale bar 10 μm. ( c ) Quantification of AmotL2 co-localization to β-catenin in control and Par3 siRNA treated MS-1 cells. N(control siRNA) = 64 cells, N( Par3 siRNA) = 42 cells, p = 8.80 × 10 −5 . ( d ) Quantification of Par3 co-localization to β-catenin in control and AmotL2 siRNA treated MS-1 cells. N(control siRNA) = 38 cells, N( AmotL2 siRNA) = 70 cells, p = 0.87, Error bars indicate standard deviation (s.d.), N.S. = Non Significant, ***p ≤ 0.001. ( e ) co-IP demonstrating the association of both Par3 and AmotL2 to the E-cadherin protein complex. Interaction of AmotL2 to E-cadherin and MAGI-1/ β-actin was dependent on Par3.

    Article Snippet: #07–330, millipore), anti-Par6 (rabbit polyclonal, #ab45394–100, abcam), anti-aPKC (anti-rabbit, #sc-216, santa cruz), anti-E-cadherin (1:500, mouse monoclonal, #610183, BD Transduction Laboratories), anti-β-catenin (1:500, #610154, 14/beta-catenin, BD Transduction Laboratories), anti-α-catenin (1:500, #610193, 5/alpha-catenin, BD Transduction Laboratories), anti-MAGI1 (1:500, # WH0009223M3, 7B4, Sigma), anti-β-actin (1:20000, #ab3280, ALTN05CC4, Abcam), Tubulin (Sigma, T5168).

    Techniques: Mass Spectrometry, Standard Deviation, Co-Immunoprecipitation Assay