Gapdh

The total protein extract was prepared by homogenizing the cells in 1 × SDS sample buffer. All the protein samples were boiled for 5 min and then resolved in SDS-PAGE. The protein samples separated by SDS-PAGE were transferred to the Immunobilon-FL PVDF membrane (Merck Millipore, IPFL00010). After blocking with 5% non-fat milk in TBST (TBS, pH 7.4, 0.1% Tween-20), the membrane was incubated with primary antibody at 4 ℃ overnight. The membrane was washed and incubated with secondary antibodies in the dark for 2 h. The image was acquired by Li-Cor Odyssey Clx Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA). The following primary and secondary antibodies were used for western blotting assay: SQSTM1 (Proteintech, 18420-1-AP), ubiquitin (Abcam, ab134953); Phospho-SQSTM1 (Thr269/Ser272)-specific antibody (Phosphosolutions, P196-269); Phospho-SQSTM1 (S403)-specific antibody (Cell Signaling Technology, 39786); FLAG tag (Prospec, ANT-146); GAPDH (Zen Bioscience, 200306); β-actin (Zen Bioscience, 200068-6D7). Dylight 680, or Dylight 800-conjugated secondary antibodies (Thermo Fisher Scientific, A28183, A27042, 35518). See Additional file 1: Table S2 for further details and dilutions of all antibodies.

The total protein extract was prepared by homogenizing the cells in a 1× SDS sample buffer. The NP-40-soluble and -insoluble protein fractions from the cells were prepared as previously described [34 (link)]. Immunoprecipitation and western blot were carried out as previously described [34 (link)]. Primary antibodies against the following proteins were used for western blot analysis: K48-linked Ub chain-specific antibody (Millipore, 05-1307); K63-linked Ub chain-specific antibody (Abcam, ab179434); ubiquitin (Abcam, ab134953); Phospho-SQSTM1 (Thr269/Ser272)-specific antibody (Phosphosolutions, P196-269); SQSTM1 (Santa Cruz Biotechnology (sc-28359); ATG5 (Cell Signaling Technology, 12994); LC3B (Cell Signaling Technology, 3868); ATG16L1 (Abcam, ab187671); WIPI2 (Abcam, ab105459); Beclin1 (Proteintech, 11306-1-AP); GFP (Rockland, 600-101-215); FLAG tag (Prospec, ANT-146-b); Myc Tag (Biolegend, MMS-150R); GAPDH (Zen Bioscience, 200306); β-actin (Zen Bioscience, 200068-6D7). See Supplementary Table S2 for further details and dilutions of all antibodies.

Protein extraction was performed with lysis buffer, radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, China) containing 1% phenylmethanesulfonyl fluoride (PMSF) (Beyotime Biotechnology, China), and subsequent sonication. After centrifugation, the supernatant was boiled for denaturation and determination of total protein concentration using the BCA protein assay kit (Beyotime Biotechnology, China). Equivalent amounts of protein were loaded for electrophoresis on 7-10% SDS-PAGE gels (Omni-Easy™ One-Step PAGE Gel Fast Preparation Kit, EpiZyme, China) and transferred to polyvinylidene fluoride membranes (PVDF, 0.2 μm, Bio-Rad). After the membranes were blocked with 5% skim milk at room temperature for 1 h, proteins were incubated overnight at 4°C with the following primary antibodies: Sox9 (Zen Bio, 1 : 2000), COL2A1 (Abcam, 1 : 3000), COL10A1 (Santa Cruz Biotechnology; 1 : 1000), Smad7 (Santa Cruz,1 : 1000), and GAPDH (Zen Bio, 1 : 2000). After the membranes were washed with TBST, they were incubated with the corresponding secondary antibodies (goat anti-rabbit IgG, 1 : 10000, Zen Bio) for 1 h at room temperature. Following sequential washing with TBST and TBS, the target proteins were detected by an ECL detection kit (Thermo Fisher Scientific), and ImageJ software was used for quantification of band density.

Immunoprecipitation (IP) and immunoblotting (IB) analyses were performed as previously described [22, 27]. Antibodies used were specific for Pin1 (rabbit polyclonal antibody; Cell Signaling Technology, Beverly, MA, USA; 1 : 1000), JNK1 (rabbit polyclonal antibody; Abcam, Cambridge, MA, USA; 1 : 1000), HA (mouse monoclonal antibody; Millipore, Billerica, CA, USA;1 : 500), p63 (rabbit polyclonal antibody; Zen‐bio, Chengdu, Sichuan, China; 1 : 1000), PARP1 (rabbit polyclonal antibody; Zen‐bio, Chengdu, Sichuan, China; 1 : 2000), and GAPDH (rabbit polyclonal antibody; Zen‐bio, Chengdu, Sichuan, China; 1 : 1000). Blots were detected using an ECL system (GE Amersham Pharmacia Biotech, Boston, MA, USA).

Real-time RT-PCR and western blotting were conducted as previously described21 (link). PCR primer sequences are shown in Supplementary Table S1. Primary antibodies against APEX1 (ab194, Abcam), DNMT1 (sc-10222, Santa Cruz), ERK (#4695, Cell Signaling Technology), P-ERK (#4370, Cell Signaling Technology), JNK (#9258, Cell Signaling Technology), P-JNK (#4668, Cell Signaling Technology), and GAPDH (200306-7E4, ZEN) were used. Images were captured with an Image Quant LAS 4000 Mini (GE Healthcare Life Sciences), and proteins on blots were quantified by scanning densitometry (ImageQuant TL, GE Healthcare Life Sciences). Experiments were performed independently for each sample, and at least three technical replicates were performed for each of the treated samples and controls.

BeWo cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (150 mM sodium chloride, 1.0% NP-40, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM pH 8.0 Tris buffer). Whole cell protein was extracted by 4°C centrifugation at 12000 g for 30 min. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Cat. No: 23227, Thermo Scientific). Protein samples (45 μg) were loaded on 12% SDS-polyacrylamide gels, resolved by electrophoresis, and transferred to polyvinylidene difluoride (PVDF) membranes (BR07146602, 0.22 mm, Merck Millipore). Immunoblotting was performed using primary antibodies against LC3 (1:1000, 12741, CST), Beclin-1 (1:1000, 3495, CST), CYP11A1 (1:1000, 14217, CST), VDAC-1 (1:1000, Proteintech), PINK-1 (1:1000, 6946, CST), P62 (1:10000, ab109012, Abcam), MMP2 (1:2000, EPR1184, Abcam), or GAPDH (1:150000, Zen Bioscience), at 4°C overnight followed by incubation with a HRP-conjugated goat anti-rabbit IgG (1:10000, ZB-2301, ZSGB-BIO) secondary antibody at room temperature (RT) for 1 hour. The blots were exposed using a chemiluminescence kit (WBKLS0500, Merck Millipore) and the signal was detected using a gel imaging system. The corresponding internal reference GAPDH was used as an internal control. The intensity of bands was analyzed using Image J (National Institutes of Health, USA).

Total proteins were extracted with RIPA Lysis (Beyotime, Shanghai, China). Equal amounts of protein were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and wet-blotted on polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Blocked in 5% non-fat milk (Biosharp, Shanghai, China) for 1 h, the membranes were incubated with primary antibodies (YAP1, 1:1000, Cell Signaling Technology, Danvers, MA, USA; p53, 1:1000, Cell Signaling Technology, Danvers, MA, USA; p21, 1:1000, Abcam, Cambridge, UK; p16, 1:2500, Abcam, Cambridge, UK; GAPDH,1:5000, ZEN-BIOSCIENCE, Chengdu, China). Protein bands were stained using HRP-conjugated secondary antibodies (goat anti-rabbit IgG-HRP, 1:5000, ZBGB-BIO, Beijing, China; goat anti-mouse IgG-HRP, 1:5000, ZBGB-BIO, Beijing, China). Membranes were developed with Plus-ECL (Bio-rad, Hercules, CA, USA) substrates and exposed to a luminescent image analyzer (Bio-OI, Guangzhou, China). Quantitative densitometry of the immunoreactive bands was performed using ImageJ.