Polyscreen pvdf transfer membrane

Western blotting was performed as described previously [88 (link)]. 1×10⁸ cells from log-phase cultures in YE media were collected. Cells were suspended in 150 μl of 10% trichloroacetic acid (TCA) and disrupted by a bead neater (TOMY, MS-100) in the presence of acid-washed glass beads. After adding 250 μl of 5% TCA, cell extracts were kept on ice for 30 min. The pellet recovered after centrifugation at 5,000 rpm for 10 min at 4°C (TOMY, Kitman) was suspended in 200 μl of SDS-elution buffer (0.5 M Tris base, 28.125 mM Tris-HCl (pH6.8), 11.25% glycerol, 0.9% sodium dodecyl sulfate, 5% β-mercaptoethanol, 0.0045% bromophenol blue) and incubated at 95°C for 2 min. The supernatant was recovered after centrifugation at 12,000 rpm for 10 min at room temperature. The cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (acrylamide to bisacrylamide ratio, 37.5:1) and transferred onto PolyScreen PVDF Transfer Membrane (Perkin Elmer, NEF1002001PK). Anti-PCNA antibodies (1:2,000) and Peroxidase AffiniPure HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 111-035-003; 1:10,000) were used as the primary and secondary antibodies, respectively. The blots were developed using Supersignal West Femto substrate (ThermoScientific, 34095). Images were acquired using ImageQuant LAS 500 (GE Healthcare).

ProteoExtract Transmembrane Protein Extraction Kit (Calbiochem) was used for protein isolation according to the manufactures protocol ‘Extraction of Membrane Proteins from Adherent Tissue Culture Cells’. Protein concentration was determined by Pierce BCA assay and 10 µg protein per lane was loaded on 12.5% polyacrylamid gels. Subsequent to SDS gel electrophoresis, proteins were blotted onto polyscreen PVDF transfer membranes (Perkin Elmer, Waltham, MA). Blots were blocked in 5% BSA before overnight incubation with primary antibodies (mouse-anti claudin-4 and -5 or rabbit-anti caudin-3, -7 and -18, all Zymed/Invitrogen, 1∶1000). Peroxidase-conjugated AffiniPure F(ab′)₂ fragment goat anti-mouse/−rabbit (1∶10000, Jackson ImmunoResearch) were used as secondary antibodies and Lumi-Light^PLUS Western Blotting Kit (Roche, Grenzach-Wyhlen, Germany) was used for visualizationin a Fusion FX 7 image acquisition system (Vilber Lourmat, Eberhardzell, Germany).

Proteins extracted from ANA-1 macrophages were electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PerkinElmer, USA). Membranes were blocked with 5% (w/v) non-fat dry milk/1% (v/v) Tween 20 in PBS for 2 h at room temperature and incubated overnight with primary antibodies of HMGB1, iNOS, Arg-1, phosphorylated PI3Kγ (p-PI3Kγ), phosphorylated (p-ERK1/2), total ERK1/2 and GADPH or β-actin. After washing, HRP labeled secondary antibodies were added for 1 h at 37 °C temperature. All the antibodies were obtained from Abcam (Abcam, Shanghai, China). Detection was performed with electrochemiluminesce (ECL) and relevant blots quantified by densitometry using the accompanying computerized image analysis program (Amercontrol Biosciences, USA).

Cardiac fibroblasts/myofibroblasts were lysed with RIPA buffer and proteinase inhibitor mixture (PMSF). Nuclear and cytoplasm protein were separated by using nuclear and cytoplasm protein extraction kit (78833; Thermo Pierce, Rockford, IL, USA), according to manufacturer's instructions. Protein concentration was assessed by Bradford assay (Bio-Rad Laboratories, Shanghai, China). Total proteins were electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PerkinElmer, Boston, MA, USA). Membranes were blocked with 5% (w/v) non-fat milk in tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 hr at room temperature and incubated overnight with primary anti-col3A1, anti-col1A1, anti-β-actin (sc-28888, sc-8784, sc-47778, respectively, Santa Cruz biotechnology Inc, Santa Cruz, CA, USA), anti-OPN (ab8448; Abcam) at 4°C, respectively. After washing, HRP-conjugated secondary antibody was added for 1 hr at 37°C. Detection was performed with enhanced chemiluminescence (ECL) and relevant blots were quantified by densitometry by using the accompanying computerized image analysis program.

For Hnf4g expression in WT and KO organoids: Organoid pellets were resuspended in 100 μl whole cell lysis buffer per well of a 6‐well plate (150 mM NaCl, 50 mM Tris pH 8, 10% glycerol, 1% NP‐40, 1 mM DTT, 1× CPI). Extracts were rotated for 1.5 h at 4°C followed by seven cycles of 30 s on and 30 s off sonication (Biorupter Pico, Diagenode). Afterward, the samples were spun at 16,200 × g for 5 min at room temperature and the supernatant was stored at −80°C. Samples were loaded on an 8% SDS–PAGE gel and afterward transferred to a nitrocellulose membrane. Membranes were incubated with primary antibody in 5% milk/TBST overnight at 4°C [anti‐beta actin 1:5,000 (Abcam, ab16039), and anti‐HNF4G 1:500 (Sigma, HPA005438)]. Afterward, membranes were incubated with secondary antibody [Polyclonal Swine Anti‐Rabbit Immunoglobulins/HRP 1:3,000 (Dako, P0399)] and imaged using chemiluminescent substrate (Thermo Fisher, 34580).
For mitochondrial ETC complexes: Organoids were washed once and Matrigel was mechanically removed in cold PBS. Total proteins were collected by direct lysis of organoids in Laemmli sample buffer. Proteins were run in SDS–PAGE and transferred to Polyscreen PVDF transfer membranes (PerkinElmer). Antibodies: Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam) and anti‐vinculin (V9131, Sigma).