Journal: Antioxidants

Article Title: CHIP Haploinsufficiency Exacerbates Hepatic Steatosis via Enhanced TXNIP Expression and Endoplasmic Reticulum Stress Responses

doi: 10.3390/antiox12020458

Figure Lengend Snippet: CHIP is involved in unfolded protein responses (UPRs) and apoptosis in hepatocytes. ( a ) AML12 cells were transfected with control (siControl) or CHIP siRNA (siCHIP) for 48 h and then treated with tunicamycin (TM, 5 or 10 μM) for 24 h. Protein levels of GRP78, ATF6, XBP-1s and CHIP were determined by immunoblotting. A-tubulin was used as a loading control. ( b ) AML12 cells were transfected with siControl or siCHIP for 48 h and then treated with TM (10 μM) for 24 h. Protein levels of cleaved PARP-1, cleaved caspase-3 (cleaved Casp3), and CHIP were determined by immunoblotting. α-tubulin was used as a loading control. ( c ) AML12 cells were transfected with siControl or siCHIP for 48 h and then treated with TM (10 μM) for 24 h. Expression of UPR-related genes were measured by qRT-PCR. Relative expression levels were normalized to GAPDH levels. ** p < 0.01 vs. siControl, ## p < 0.01 vs. siCHIP, † p < 0.05 and †† p < 0.01. ( d ) Primary hepatocytes from CHIP +/+ , CHIP +/− , and CHIP −/− mice were treated with TM (2 or 10 μM) for 6 h. Protein levels of GRP78, CHOP, XBP-1s, and CHIP were measured by immunoblotting. β-actin was used as a loading control. ( e ) Primary hepatocytes from CHIP +/+ and CHIP +/− mice were treated with brefeldin A (BFA, 1 or 2 μM) for 6 or 9 h. Protein levels of GRP78, CHOP, cleaved PARP-1, and CHIP were measured by immunoblotting. α-tubulin was used as a loading control.

Article Snippet: The antibodies were purchased from the following vendors: TXNIP (MBL International, Woburn, MA, USA); KDEL (GRP94, GRP78) (Enzo Life Sciences, Lörrach, Germany); XBP-1s (BioLegend, San Diego, CA, USA); ATF6 (Novus Biologicals, Littleton, CO, USA); ATF4, GADD153 (CHOP), HA, and CHIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Akt, p-Akt, ACC, FAS, PARP-1, and cleaved Caspase-3 (Cell Signaling, Danvers, MA, USA); NLRP3 (ThermoFisher, Waltham, MA, USA); PGC1α (abcam, Cambridge, UK); and α-tubulin and β-actin (Sigma Aldrich, St. Louis, MO, USA).

Techniques: Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR, ChIP-chip

Journal: Journal of Experimental & Biomedical Sciences/Biomedical Science Letters

Article Title: Expression of Beta-catenin-interacting Protein 1 (CTNNBIP1) Gene Is Increased under Hypothermia but Decreased under Additional Ischemia Conditions

doi: 10.15616/bsl.2014.20.3.168

Figure Lengend Snippet: Fig. 3. Effect of hypothermia on gene expression of ER chaperones (A), XBP1 mRNA splicing (B), ER stress sensors (C), apoptosis- associated factors (D) and autophage-associated factors (E). PC12 cells were incubated under control conditions (37 ) or hypothermia ℃ (32 ) for 1 day. Gene expressions were determined by RT ℃ -PCR by conditions described in Materials and methods (A, D, E). The product of XBP1-Pst1 digestion was subjected to electrophoresis on the agarose gel (B). Both cleavage of ATF-6 and phosphorylation elF2α were assessed by Western blotting (C). The experiments were performed thrice and results represent the average and representative findings are shown. Bip; binding immunoglobulin protein, Canx; calnexin, PDI; protein disulfide isomerase, ATF6; activating transcription factor 6, phosph-elF2α; phosphorylation of eIF2α, Bak1; BCL2-antagonist/killer 1, Bcl-2; B-cell lymphoma 2, LC3; microtubule-associated protein 1 light chain 3α, beclin; coiled-coil myosin-like BCL2-interacting protein.

Article Snippet: Mouse anti-ATF6 antibody was obtained from Imgenex (San Diego, CA, USA).

Techniques: Gene Expression, Incubation, Control, Electrophoresis, Agarose Gel Electrophoresis, Phospho-proteomics, Western Blot, Binding Assay