Journal: Scientific Reports
Article Title: Bcl2 is a critical regulator of bile acid homeostasis by dictating Shp and lncRNA H19 function
Figure Lengend Snippet: Bcl2-mediated SHP protein degradation is associated with activation of caspase 8 pathway. ( A ) Micrographs of DAPI staining (blue), MitoTracker staining (red), and GFP-SHP expression (green) in Huh7 cells transfected with GFP-SHP plasmid, alone or in combination with wide-type Bcl2 (Bcl2wt) or Bcl2 lacking the TM domain (Bcl2ΔTM). Magnification of 20X. ( B ) RT-PCR of Shp and Bcl2 mRNAs in various cells. ( C ) Western blot of SHP protein in Huh7 cells that were expressed with Bcl2 or Bcl2ΔTM in the absence or presence of protein synthesis inhibitor cycloheximide (CHX). ( D ) Left: Western blot of SHP and BCL2 proteins in Huh7 cells. SHP protein was detected by anti-Flag or anti-SHP antibody. Middle: Western blot of SHP and BCL2 proteins in the liver. The mice were the same as in Fig. 1 . Left: Western blot of SHP and BCL2 proteins in primary mouse hepatocytes. The corresponding mRNA level is in Fig. S4A . Hepatocytes infected with ade-SHP served as a positive control. ( E ) Left and middle: Western blot of SHP and BCL2 proteins in HepG2 cells treated with DMSO control or 5 μM MG132 for 6 hr. The endogenous SHP protein and exogenously expressed Flag-SHP protein were detected by anti-SHP or anti-Flag antibody, respectively. Right : Western blot of BCL2 and cas-3 proteins in Bcl2 overexpressed liver. ( F ) Western blot of SHP and BCL2 proteins in HEK 293T cells. Left: Cells were pre-treated with caspase inhibitors (50 μM) for 1 hr followed by plasmid transfection for 24 hr. Right: Cells were pre-treated with various inhibitors for 1 hr followed by plasmid transfection for 24 hr. For TNFα+ CHX and staurosporine groups, cells were transfected with plasmids for 20 hr followed by a 4 hr treatment. Dose: SP600125 (SP) 50 μM, Rapamycin (Rapa) 10 μM, LY294002 (LY) 50 μM, U0126 10 μM, TNFα 50 ng/ml + CHX 50 μM, staurosporine (STS) 1 μM, and Z-VAD 50 μM.
Article Snippet: The following antibodies were used for protein precipitation (IP) and Western blots (WB): antibodies against Flag (Sigma, F7425), GFP (Sigma, G1544), Bcl2 (Abcam, ab7973), β-actin (Sigma, A-1978), α-Tubulin (Sigma, T6074), PARP (Cell signaling, #9542), SHP (Santa Cruz, sc-30169), caspase-3 (Cell signaling, #9661), cleaved caspase-8 (Cell signaling, #9748).
Techniques: Activation Assay, Staining, Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mouse Assay, Infection, Positive Control