Lgals1

Samples were sectioned at a thickness of 5 µm. All the sections were incubated for 1 hour with primary antibody directed against IL1B (Cell Signaling Technology, #2022, 1:200), COL2A1 (Santa Cruz, sc-52658, 1:200), COL4A1 (Thermo Fisher Scientific, PA5-85634, 1:500), LGALS1 (Cell Signaling Technology, #5418, 1:200), MMP3 (Abcam, ab223666, 1:500) after blocking endogenous peroxidase using 3% hydrogen peroxide for 5 minutes at room temperature. After rinsing, the sections were incubated for 1 hour with biotinylated horseradish peroxidase-conjugated goat antirabbit IgG. Diaminobenzidine was used to develop peroxidase staining.

For coimmunoprecipitations, 300 µg of starting protein was used per sample. Extract was precleared with mouse IgG conjugated resin (30 µL of 50% slurry) for 30 min at 4°C. Samples were then incubated with 3 µg of antibody for 1.5–2 h at 4°C. Forty-five micrograms of Protein G plus Protein A resin was added and incubated for another hour. For Flag IPs, 30 µL of a 50% slurry of Flag conjugated resin was added to the extract after preclear and incubated for 2.5–3 h. Samples were washed 6× with IP₃₀₀ Buffer (20 mM Tris–HCl, pH 7.4, 2 mM MgCl₂, 300 mM NaCl, 0.05% NP-40) with 5 min incubations and eluted with 95°C 1×SDS for 5 min with shaking. Samples were loaded onto 4%–20% SDS-PAGE gels and Western blots were performed. Antibodies used: GW182 (Bethyl, A302-329A); DICER (Abcam, ab14601); TRBP (Abcam, ab72110); AGO1 (Cell Signaling, D84G10); AGO3 (Sigma, SAB4200112); KPNB1 (Bethyl, A300-482A); PTBP1 (Abcam, ab5642); LGALS1 (Cell Signaling, 5418S); PSPC1 (Santa Cruz, SC84576); SMARCC1 (Abcam, ab72502).