Western lightning enhanced chemiluminescence

Intestinal organoids were pooled from 3 wells (from 24-well plate), washed to remove matrigel and homogenized in Pierce RIPA Lysis and Extraction Buffer (ThermoFisher, cat # 89900), with freshly added Halt Protease Inhibitor Cocktail (100X) (ThermoFisher cat #78430). Crude lysates were centrifuged at 14,000g for 15 min and protein concentrations determined using Bio-Rad protein assay reagent. Samples were diluted in SDS sample buffer. Protein concentrations were measured by BCA (bicinchoninic acid assay) method. Bound proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad cat #170–4159). Individual proteins were detected with specific antibodies and visualized on film using horseradish peroxidase–conjugated secondary antibodies (Bio-Rad) and Western Lightning enhanced chemiluminescence (PerkinElmer Life Sciences). Antibodies to Phospho-Histone H2AX (Ser139) or γH2AX (20E3) Rabbit mAb (cat #9718), H2AX (cat #2595), were purchased from Cell Signaling and used at dilutions recommended by the manufacturer.

We homogenized tissues in lysis buffer (50 mM Tris pH 7.5, 150 mM NaC1, 2mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, 1 mM Na₃VO₄, 5 mM NaF, 1 mM phenylmethanesulfonylfluoride, 25 mM glycerol 2-phosphate and freshly added protease inhibitor tablet) and then incubated them for 1 h at 4 °C. Cell lysates were produced in an SDS lysis buffer (100 mM Tris pH 7.5, 130 mM NaC1, 1% NP-40, 0.1% SDS, 0.2% sodium deoxycholate, 1 mM Na₃VO₄, 1 mM NaF, 100 mM Na₄P₂O₇, l mM phenylmethanesulfonylfluoride, 25 mM glycerol 2-phosphate and freshly added protease inhibitor tablet) and sonicated for three 5 s pulses at an output power of 6. We centrifuged crude lysates at 14,000 g for 15 min twice and determined the protein concentration using Bio-Rad Protein Assay Dye Reagent. Samples were diluted in SDS sample buffer. Bound proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Bio-Rad). Individual proteins were detected with specific antibodies and visualized on film using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) and Western Lightning Enhanced Chemiluminescence (Perkin Elmer Life Sciences).

MACS-isolated FAPs from tibialis anterior muscle were lysed at 4°C using RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, protease and phosphatase inhibitors (Calbiochem and Roche, respectively). Cell lysates were cleared by centrifugation at 16,000 g for 10 min, and protein concentration was determined using a BCA assay (Thermo Fisher Scientific). The cell lysate was denatured at 100°C for 5 min in 6X SDS-PAGE loading buffer. 5–10 µg of cell lysates were resolved on 4–15% TGX gradient gels (Bio-Rad Laboratories) and transferred to nitrocellulose membrane (Amersham). Membranes were blocked with 5% non-fat dried milk in TBS with 0.1% Tween, and then incubated with primary antibody overnight at 4°C. Primary antibodies used were mouse anti-GLI3 (1:1,000; gift of Suzie Scales, Genentech) and mouse anti-αTUB (1:5000; Sigma #T5168). After washing in TBS with 0.1% Tween, membranes were incubated in HRP-conjugated secondary antibody for 2hr at room temperature. Blots were developed with Western lightning enhanced chemiluminescence (PerkinElmer), and molecular weights were determined using full-range rainbow pre-stained protein standards (GE Healthcare).

Tissues were homogenized in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM DTT, 1 mM Na₃VO₄, 5 mM NaF, 1 mM phenylmethanesulfonylfluoride, 25 mM glycerol 2-phosphate and freshly added protease inhibitor tablet) and then incubated them for 1 h at 4 °C. Cell lysates were produced in an SDS lysis buffer (100 mM Tris pH 7.5, 130 mM NaCl, 1% NP-40, 0.1% SDS, 0.2% sodium deoxycholate, 1 mM Na₃VO₄, 1 mM NaF, 100 mM Na₄P₂O₇, 1 mM phenylmethanesulfonylfluoride, 25 mM glycerol 2-phosphate and freshly added protease inhibitor tablet) and sonicated for three 5 s pulses at an output power of 6. Crude lysates were centrifuged at 14,000 g for 15 min twice and the protein concentration was determined using Bio-Rad Protein Assay Dye Reagent. Samples were diluted in SDS sample buffer. Bound proteins were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Bio-Rad). Individual proteins were detected with specific antibodies and visualized on film using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) and Western Lightning Enhanced Chemiluminescence (Perkin Elmer Life Sciences). TBK1 (3013), phosphorylated TBK1 (Ser172–5483), p38 (9212), and phosphorylated p38 (Thr180/Tyr182–9211)-specific antibodies were purchased from Cell Signaling Technology (Danvers, MA).