Ab170901

The xenograft tumor or lungs were dissected, fixed, and paraffin-embedded. The sections were stained with H&E [27 (link)]. For immunohistochemistry (IHC) analysis, the slides were stained with anti-PD-L1 (1:200, ab205921, Abcam), Ki-67 (1:200, ab15580, Abcam), SYVN1 (1:100, ab170901, Abcam), and FoxO1 (1:100, ab39670, Abcam) at 4 °C overnight. The slides were then incubated with secondary antibody. The signal was detected using the DAB substrate (Beyotime).

Ginsenoside Rg1 (purity of 98%) was purchased from manster biotechnology co., Ltd. (Chengdu, China). Doxorubicin (a purity of 98%) was procured from Aladdin Industrial Company (Shanghai, China). Antibodies against LC3A/B (4108), P70S6K (2708), p-P70S6K (9234), P62 (23214) and Beclin1 (3495) were purchased from Cell Signaling Technology (Shanghai, China). Antibody against ATF6 (sc-22799), XBP1 (sc-7160) and GFAT1 (sc-134894) were purchased from Santa Cruz Biotechnology (Shanghai, China). Antibodies against IRE1 (ab37073), ATG5 (ab108327), TIF1 (ab174287), GRP78 (ab108615), HRD1 (ab170901), FAM134B (ab151755), JNK1 (ab179461) and GAPDH (abb181602) were purchased from Abcam (Shanghai, China).

The total protein of HBMECs was extracted with RIPA buffer and 1% PMSF (Beyotime, Shanghai, China) for Western Blot. The concentration of proteins was measured by using a Pierce BCA protein assay kit (Beyotime, Shanghai, China). The proteins in the extracts were separated by 10% SDS-PAGE gels, and then were transferred from SDS-PAGE to PVDF membranes. After sealing with 5% skim milk for 1 hour, the membranes were incubated with the primary antibodies overnight at 4°C. After washing with TBST for three times, the membranes were incubated with the secondary antibodies for 1.5 hours. Finally, a chemiluminescence detection system was used to observed protein samples. The antibodies were used as follow: anti-NRF2 (68 kDa, 1 : 1000, ab62352, Abcam); anti-KEAP1(70 kDa, 1 : 1000, ab139729, Abcam); anti-HRD1 (67 kDa, 1 : 2000, ab170901, Abcam); anti-MCM2 (102 kDa, 1 : 2000, ab108935, Abcam); and anti-β-actin (42 kDa, 1 : 1000, sc-47,778, Santa Cruz).

The details of these procedures were described previously [37 (link)]. Anti-SIX3 (Santa Cruz Biotechnology, Dallas, TX, USA; sc-81985), anti-FLAG (Abcam, Cambridge, MA, USA; ab205606), anti-HA (Abcam; ab9110), and anti-IgG (Santa Cruz Biotechnology; sc-2027) antibodies were used for co-immunoprecipitation (Co-IP). Immunoprecipitates were washed at least five times and subjected to western blot analysis using anti-TRIM27 (Abcam; ab78393), anti-NEDD4 (Abcam; ab236512), anti-SMURF2 (Abcam; ab94483), anti-RNF6 (Abcam; ab204506), anti-SYVN1 (Abcam; ab170901), anti-MDM2 (Abcam; ab16895), and Anti-SIX3 (Abcam; ab172131) antibodies. For ubiquitination assays, the lysates of A549 cells transfected with siTRIM27-1 or siNC were used for IP with an anti-IgG (Santa Cruz Biotechnology; sc-2027) or Anti-SIX3 antibody (Santa Cruz Biotechnology; sc-81985) and Protein A/G PLUS-Agarose (Novex, Oslo, Norway), which was performed at 4° C overnight. The eluted proteins were then detected by western blot analysis using an anti-ubiquitin (Ub) antibody (Abcam, ab7780).

To obtain total cellular protein, cells and rat ovaries were treated with RIPA buffer containing protease inhibitors. The total protein was further quantified using the BCA method (Pierce, Rockford, IL, USA). A 10% SDS–polyacrylamide gel was used to separate 20 μg of total protein by electrophoresis. The proteins were transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were further blocked with 5% nonfat milk and subsequently incubated with rabbit monoclonal SYVN1 antibody (Abcam, ab170901; 1 : 1,000), rabbit polyclonal cleaved-caspase-3 (Abcam, ab49822, 1 : 500), rabbit polyclonal Bax (Sigma-Aldrich, B3428, 1 : 500), rabbit polyclonal Bcl-2 (Abcam, ab196495, 1 : 500), mouse monoclonal Drp1 (Abcam, ab156951, 1 : 1,000), and rabbit polyclonal Mnf1 (Sigma-Aldrich, SAB2106161, 1 : 1,000), overnight at 4°C. Next, the membranes were incubated with respective peroxidase-conjugated secondary antibody for 1 h. Proteins bands were finally detected using an enhanced chemiluminescent detection system (Immunoblot, 23225; Millipore, Billerica, MA, USA). Band intensity of the target protein was obtained using ImageJ (NIH, USA) and normalized with that of the mouse monoclonal β-actin (Abcam, ab8224, 1 : 1,000).