Journal: Experimental and Therapeutic Medicine

Article Title: Effects of celecoxib on cell apoptosis and Fas, FasL and Bcl-2 expression in a BGC-823 human gastric cancer cell line

doi: 10.3892/etm.2017.4769

Figure Lengend Snippet: Fas, FasL and Bcl-2 protein expression in a BGC-823 gastric cancer cell line treated with various concentrations of celecoxib for 48 h (n=3; mean ± standard deviation).

Article Snippet: Rabbit anti-human GAPDH (catalogue no. BA2913), Fas (catalogue no. PB0214), FasL (catalogue no. BA0049) and Bcl-2 (catalogue no. BA0412) monoclonal antibodies and goat anti-rabbit antibodies (catalogue no. BA1055) labeled with horseradish peroxidase were purchased from Boster Biological Technology, Ltd., (Wuhan, China).

Techniques: Expressing

Journal: PLoS Genetics

Article Title: The prefoldin complex stabilizes the von Hippel-Lindau protein against aggregation and degradation

doi: 10.1371/journal.pgen.1009183

Figure Lengend Snippet: A) Scheme depicting the different BirA fusion proteins used in the BioID experiment (ex1, ex2 and ex3 refer to VHL exon1, exon2 and exon3-encoded polypeptides, respectively), B) Western blot analysis of PFDN1, PFDN2, PFDN3 and BirA fusion proteins in total protein extracts before (Input) and in fractions recovered after the Streptavidin affinity-chromatography column (Bound). Cullin 2 (CUL2) and Elongin C (ELOC) were used as positive controls whereas GAPDH and p44/42 ERK were used as negative controls. Ctl corresponds to untransfected control cells. Left: HEK293 cells and right HeLa cells. C) Quantification of the biotinylated prefoldin / pVHL expression levels to map the prefoldin binding site in pVHL ORF: truncated VHL213-BirA constructs expressing either all 3 exons (VHL 213 ), exons 1+3 (VHL 172 ), exons 1+2 (VHL ex1&2 ) and exons 2+3 (VHL ex2&3 ) were used in a BioID experiment in HeLa and 786-O cells. Histograms represent the mean relative levels of recovered PFDN1 (upper panel) and PFDN3 (lower panel) proteins. Full-length VHL213-BirA level was set as 1. D) HeLa cells expressing GFP, VHL213-GFP (213), VHL172-GFP (172) or control untransfected cells (Ctl) were lysed and immunoprecipitation was performed with GFP-trap. The whole cell lysates (Input) and immunoprecipitates (Bound) were analyzed by Western blot using anti-GFP, anti-CUL2, anti-PFDN1, anti-PFDN3 and anti-PFDN5 antibodies.

Article Snippet: Unless otherwise specified, membranes were blocked with 5% non-fat dry milk in TBS/0.1% Tween 20 (TBST/milk) and incubated overnight at 4°C with the following primary antibodies diluted in TBST/milk: rabbit monoclonal α-cullin 2 (Invitrogen; 1:500), rabbit polyclonal α-Elongin C (BioLegend; 1:1000), rabbit polyclonal α-ERK 1/2 (Santa Cruz Biotechnology; 1:3000), rabbit polyclonal GAPDH (Cusabio; 1:3000), mouse monoclonal α-GFP (Roche; 1:4000), rat monoclonal α-HA (Roche; 1:4000), rabbit monoclonal p21 WAF1/CIP1 (Cell Signaling Technology; 1: 1000), rabbit polyclonal α-Prefoldins 1, 2, 4 and 5 (Abclonal; 1:1000–1:3000), mouse monoclonal α-prefoldin3/VBP1 (Santa Cruz Biotechnology; 1:250), mouse monoclonal α-PSTAIR (Sigma; 1:5000), mouse monoclonal α-ubiquitin (P4D1; Santa Cruz Biotechnology; 1:1000), mouse monoclonal α-HSP70 (Abcam; 1:1000) and mouse monoclonal α-VHL [ ] (1:1000).

Techniques: Western Blot, Affinity Column, Control, Expressing, Binding Assay, Construct, Immunoprecipitation

Journal: PLoS Genetics

Article Title: The prefoldin complex stabilizes the von Hippel-Lindau protein against aggregation and degradation

doi: 10.1371/journal.pgen.1009183

Figure Lengend Snippet: A) Western blot analysis of PFDN1, PFDN2, PFDN3, PFDN4 and PFDN5 expression levels after siRNA targeting PFDN1, PFDN3 or PFDN1+PFDN3 in HEK293 cells. GAPDH was used as a loading control. B) Histogram representing the amount of PFDN subunits levels in siRNA experiments (mean±s.d. from at least three independent experiments). “Si Ctl” represents cells transfected with mock siRNA. C) Up: Western blot analysis of pVHL213 expression after siRNA of PFDN1, PFDN3 or PFDN1+PFDN3 in HEK293 cells. Bottom: Histogram representing the pVHL213 levels in PFDN siRNA experiments (mean±s.e.m. from at least three independent experiments). *, p-value<0.05; **, p-value<0.01. Mann-Whitney test.

Article Snippet: Unless otherwise specified, membranes were blocked with 5% non-fat dry milk in TBS/0.1% Tween 20 (TBST/milk) and incubated overnight at 4°C with the following primary antibodies diluted in TBST/milk: rabbit monoclonal α-cullin 2 (Invitrogen; 1:500), rabbit polyclonal α-Elongin C (BioLegend; 1:1000), rabbit polyclonal α-ERK 1/2 (Santa Cruz Biotechnology; 1:3000), rabbit polyclonal GAPDH (Cusabio; 1:3000), mouse monoclonal α-GFP (Roche; 1:4000), rat monoclonal α-HA (Roche; 1:4000), rabbit monoclonal p21 WAF1/CIP1 (Cell Signaling Technology; 1: 1000), rabbit polyclonal α-Prefoldins 1, 2, 4 and 5 (Abclonal; 1:1000–1:3000), mouse monoclonal α-prefoldin3/VBP1 (Santa Cruz Biotechnology; 1:250), mouse monoclonal α-PSTAIR (Sigma; 1:5000), mouse monoclonal α-ubiquitin (P4D1; Santa Cruz Biotechnology; 1:1000), mouse monoclonal α-HSP70 (Abcam; 1:1000) and mouse monoclonal α-VHL [ ] (1:1000).

Techniques: Western Blot, Expressing, Control, Transfection, MANN-WHITNEY