Ab41898

TRAF3 expression and localization in the liver sections of mice were investigated using TRAF3 and HNF4 (marker of hepatocytes) co-staining63 (link). The mouse HNF4-specific antibody (ab41898, Abcam) and the rabbit anti-mouse TRAF3 antibody (ab76147; Abcam) were used as primary antibodies, and goat anti-mouse IgG and anti-rabbit IgG (Invitrogen) antibodies were applied as the corresponding secondary antibodies. Immunofluorescence images were obtained using fluorescence microscope (Olympus) running DP2-BSW software (version 2.2).

Five µm sections from FFPE samples were stained overnight using antibodies to Huntingtin (Millipore MAB2166; mouse monoclonal, clone 1HU-4C8, 1∶150), HNF4α (AbCam ab41898; mouse monoclonal, clone K9218, 1∶70), 14-3-3ζ (AbCam ab51129; rabbit polyclonal, 1∶50) and c-Myc (AbCam ab32072; rabbit monoclonal, clone Y69, 1∶100). Antigen retrieval was performed using hot citrate buffer pH 6.0 (Huntingtin, HNF4α, and 14-3-3ζ) or 1 mmol/L EDTA pH 8.0 (c-Myc). Antigen-antibody reactions were visualized using a polymer-based detection system (EnVision Kit, Dako), using diaminobenzidine as chromogen.

Liver tissues were immobilized with 4% paraformaldehyde (PFA), dehydrated, embedded in paraffin, sectioned at 5 μm, and processed for immunofluorescent staining. SOX9 antibody (AB5535, Millipore, USA) and HNF4α antibody (Ab41898, Abcam, Cambridge, England) were used to incubate sections at 4°C overnight. Slides were washed with PBS and incubated with corresponding secondary antibodies for 1 h, followed by PBS washes. The incubated slides were added with DAPI for nuclear staining and mounting. Images were acquired with a laser scanning confocal microscope (LSM710, Carl Zeiss Microscopy), were analyzed by Zen software with fixed parameters.

Left ventricular myocardial tissue or cardiomyocytes were isolated and lysed with cold 1 × lysis buffer supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and then mixed with 1 × SDS sample buffer and boiled at 95 °C for 5 min. The lysate was resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked and incubated with primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. The signals were detected using the ECL system (Pierce) and quantified by scanning densitometry using the Image Lab analysis system (see Supplementary Material for original western blots). The membranes were stripped with stripping buffer (P0025, Beyotime, Beijing, China) and reblotted with next primary antibody. Primary antibodies: anti-Peli1 (ab199336, Abcam, Cambridge, MA, USA), anti-HNF4α (ab41898, Abcam, Cambridge, MA, USA), anti-GAPDH (AF0006, Beyotime, Beijing, China), anti-Flag (F2555, Sigma-Aldrich, St. Louis, MO, USA), anti-Myc (AF2864, Beyotime, Beijing, China), anti-His (AF2876, Beyotime, Beijing, China), anti-HA (AF2858, Beyotime, Beijing, China).

Liver tissues were lysed in Radio-Immunoprecipitation Assay lysis buffer (MA0151, Dalian Meilun Biotechnology co., Ltd., Dalian, China), and then centrifuged at 13,680 x g, 4°C, 30 min, and then the supernatant was harvested. Protein concentration was measured via BCA kit (P0011, Beyotime, Shanghai, China). After that, equal amounts of protein (30 μg) were separate via the SDS-PAGE, and subsequently transferred to the PVDF membrane. The PVDF membrane was blocked with 5% skimmed milk (0040895, Biosharp, Hefei, China) in TBST buffer for 1 hour at room temperature, then incubated with primary antibodies in 4°C for overnight, and then incubated with HRP (horseradish peroxidase)-labeled secondary antibodies, the signals were detected using enhanced chemiluminescence reagent. The primary antibodies included: rabbit anti-GAPDH (1:2,500; ab9485; Abcam), rabbit anti-CD36 (1:1000; ab133625; Abcam), mouse anti-HNF4a (1:5,000; ab41898; Abcam), rabbit anti-HMGCR (1:1,000; ab174830; Abcam), mouse anti-SCD1 (1:1,000; ab19862; Abcam), rabbit anti-FASN (1:1,000; #3180; CST). Secondary antibodies included HRP-goat anti-rabbit IgG (1:5,000; os0701; Earthox Life Sciences) and HRP-donkey anti-mouse IgG (1:2,000; ab150105; Abcam). The quantification of WB bands was analyzed through the Lane 1d software (version 5.1.0.0; SageCreation).