Igg control antibody

Western blotting was performed as described previously using specific antibodies against α-SMA (ab7817, Abcam, 1:5000), TGFβ1 (sc-130348, Santa Cruz Biotechnology, 1:1500), Bnip3 (ab109362, Abcam, 1:7000), phospho-DRP1 (Ser616) (#4494, Cell Signaling Technology, 1:2000), Drp1 (#8570, Cell Signaling Technology, 1:2000), Fis1 (ab71498, Abcam, 1:2000), RORα (sc-6062, Santa Cruz Biotechnology, 1:2000), and actin (sc-1616, Santa Cruz Biotechnology, 1:2000). The ChIP assay was performed using anti-RORα (sc-6062, Santa Cruz Biotechnology), anti-p300 (sc-585, Santa Cruz Biotechnology), and anti-histone 3 (acetyl K27) (ab4729, Abcam) antibodies or a control IgG antibody (Santa Cruz Biotechnology), and specific primers (Supplementary Table)^{19 (link)}. Relative mRNA expression was determined by qRT–PCR using the ABI StepOnePlus^TM Real-Time PCR system (Applied Biosystems, Foster City, CA) using specific primers (Supplementary Table). The mRNA expression of genes was calculated relative to controls using the 2^−ΔΔCT method^{19 (link)}.

Dulbecco’s modified Eagle’s medium (DMEM), F-12 (1:1), and bovine serum (BS) were purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Corning (Corning, NY). Melatonin, doxorubicin, H2DCF-DA, intracellular ATP assay kits, N-acetyl cysteine, and 4-P-PDOT (4-phenyl-2-propionamidotetralin) were purchased from Sigma-Aldrich (St. Louis, MO). MTT thiazolyl blue and D-luciferin were purchased from Duchefa Biochemie (Haarlem, the Netherlands). 4’,6-Diamidino-2-phenylindole (DAPI), enhanced chemiluminescence reagent, antibodies against GAPDH, E2F1, c-Myc, and voltage-dependent anion channel (VDAC), MitoTracker red, and control IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-AMPKα2 and anti-AMPKα1 antibodies were purchased from R&D Systems (Minneapolis, MN). MitoTracker green and anti-phospho-H2A.X (S139), anti-phospho-AMPKα (Thr172), anti-phospho-acetyl-CoA carboxylase (ACC) (S79), anti-ACC, anti-phospho-mitochondria fission factor (MFF) (S146), and anti-MFF antibodies were purchased from Cell Signaling Technology (Danvers, MA). MitoSOX Red was purchased from Invitrogen (Carlsbad, CA). The FITC-annexin V apoptosis kit was purchased from DB BioScience (San Diego, CA). The Total OXPHOS Rodent WB Antibody Cocktail was purchased from Abcam (Cambridge, MA).

Human fetal lung fibroblast (MRC5) cell lines were propagated in EMEM media (Gibco, Rockville, IL, USA) with 10% FBS (Hyclone, Logan, UT, USA), and 1% penicillin/streptomycin antibiotic mix (Lonza, Allendale, NJ, USA). The cells were kept in a 37 °C humidified incubator with 5% carbon dioxide. Immobilized protein A/G beads, FN, α-SMA, TβRI, HA tag, USP11, V5 tag, TβRII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-SMAD2, phospho-SMAD3, total SMAD2, SMAD3, and ubiquitin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Bleomycin, leupeptin, CHX, MTX, and antibodies against Flag-tag and β-actin were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TGF-β1 was purchased from Invitrogen (Carlsbad, CA, USA). Proteasome inhibitor MG-132 was from Calbiochem (KGaA, Darmstadt, Germany). DAPI was purchased from ThermoFisher Scientific (Waltham, MA, USA). All materials in highest grades used in the experiments are commercially available.

The cells were crosslinked with 1% formaldehyde for 10 min at 37 °C to cross-link the nuclear proteins to DNA. Subsequently, the cells were harvested by centrifugation at 4 °C for 4 min at 1000 × g, and then lysed in 200 μl SDS lysis buffer (1%SDS, 10 mM EDTA and 50 mM Tris-HCl), sheared with a Diagenode Bioruptor to chromatin fragment sizes of 200–1000 base pairs. Chromatin was immunoprecipitated with Bcl6, NcoR2, HDAC3, H3K9ac, H3K27ac or control IgG antibodies (Santa Cruz Biotechnology)56 (link). After complete washing, the immunoprecipitated DNA was quantified with real-time PCR. The IRF7-specific primer sequences are as follows: forward 5′-ATCTTGCGCCAAGACAATTCAGGG-3′ and Reverse 5′-TTGTGGCACTGCTCACCAGTAGAT-3′. The data was normalized to input (input %) or to Ig control (enrichment fold). ΔCt (normalized to the input) = (Ct [ChIP] − (Ct [Input] − Log2 (Input Dilution Factor). ΔCt (normalized to Ig) = (Ct [ChIP] − (Ct [Ig]). The relative expression levels were determined by applying the 2-ΔΔCt method.

In brief, total proteins were extracted from NT‐2 cells with Western and IP lysis buffer (Beyotime Biotech) and precleared with 30 μl protein A/G magnetic beads (Selleck) at 4°C. Meantime, 50 μl magnetic beads, mixed with 3 μg anti‐Flag antibodies (Sigma, F2555) or control IgG antibodies (Santa Cruz, sc‐2025), were pre‐incubated for 4 h at 4°C before immunoprecipitated with total proteins at 4°C overnight. Then beads were washed with Washing buffer (Beyotime Biotech, China) three times for 15 min, and resuspended in 50 μl lysis buffer with 1×SDS loading buffer, and boiled for 10 min.