Rabbit anti sqstm1 p62

Cells were lysed in RIPA lysis buffer in the presence of protease-inhibitors (Roche). Following electrophoresis using NuPAGE 4–12% gels (Thermo Fisher Scientific), proteins were transferred onto 0.45 μm nitrocellulose membranes and the membranes were incubated overnight at 4°C with the indicated primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. The following antibodies were used in this study: mouse anti-LAMP1 (Santa Cruz, sc-20011), rabbit anti-LAMP2A (Abcam, ab18528), rabbit anti-actin (Sigma, A2066), mouse anti-Rab11 (Thermo Fisher Scientific, MA1-24919), rabbit anti-SQSTM1/p62 (Cell Signaling, 5114), rabbit anti-LC3B (Cell Signaling, 2775), and goat anti-Megalin (Santa Cruz, sc-16476).

The western blot procedure was carried out as previously described [25 (link)]. Briefly, RA-FLS were washed twice with cold PBS and lysed in RIPA lysis buffer containing 50 mM Tris, 0.15 M NaCl, 1 mM EGTA, 1% NP40, 0.25% SDS (Beyotime Institute of Biotechnology, Haimen, China), protease inhibitor mix, and phosphatase inhibitors (Roche Diagnostics, Switzerland). Samples were boiled and resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, USA). Membranes were immunoblotted with primary antibodies followed by incubation with the horseradish peroxidase- (HRP-) conjugated secondary antibodies. The protein bands were visualized with the Pierce ECL Plus western blotting substrate (Thermo Fisher Scientific, Inc., USA), and GAPDH was used as the loading control. The following antibodies were used in the present study: rabbit anti-YAP (no. 14074), rabbit anti-TAZ (no. 4883), rabbit anti-ULK1 (no. 8054), rabbit anti-LC3B (no. 3868), rabbit anti-SQSTM1/p62 (no. 39749), rabbit anti-β-catenin (no. 8480), rabbit anti-Vimentin (no. 5741), HRP- linked anti-rabbit IgG (no. 7074), anti-mouse IgG (no. 7076) purchased from Cell Signaling Technology (Danvers, MA, USA), and mouse anti-GAPDH purchased from KangChen Bio-tech (Shanghai, China).

Protein lysates (12.5–50 µg for cytosolic fractions and 10–15 µg for nuclear fractions) in 1X SDS/β-mercaptoethanol buffer were resolved on a 4–20% TGS stain free gel (BioRad, Hercules, CA, USA) and electrotransferred onto a polyvinylidene difluoride transfer membrane. Western blot analysis was performed using mouse anti-lamin A/C (1:2000; Cell Signaling Technology #4777, Danvers, MA, USA), rabbit anti-SQSTM1/p62 (1:1000; Cell Signaling Technology #5114) or rabbit anti-GAPDH (1:1000; Cell Signaling Technology #5174) and horseradish peroxidase-conjugated goat anti-rabbit (1:2000; Cell Signaling Technology #7074) or goat anti-mouse (1:5000, Abcam #ab6789, Waltham, MA, USA) antibodies. Protein bands were visualized by chemiluminescence using a ChemiDocTouch Imaging System (BioRad, Hercules, CA, USA). Bands were quantified using the ImageLab software version 5.2.1.

Primary antibodies used in western blotting and immunostaining were as follows: mouse anti-cytochrome c (BD Biosciences, 556433); rabbit anti-phosphorylated H2A.X (Ser139) (Cell Signaling Technology, 9718); rabbit anti-human GAPDH (Trevigen, 2275-PC-100); rabbit anti-cleaved PARP (Cell Signaling Technology, 5625), rabbit anti-phospho-p53 (Cell Signaling Technology, 9284), mouse anti-TP53/p53 (Santa Cruz Biotechnology, SC-126), mouse anti-p21 Waf1/Cip1 (Cell Signaling Technology, 2946), rabbit anti-LC3 (MBL International, PM036), rabbit anti-SQSTM1/p62 (Cell Signaling Technology, 5114), anti-ATG3 (MBL International, M133-3), rabbit anti-Mcl-1 (Cell Signaling Technology, 5453), rabbit anti-phospho-histone H3 (Ser 10) (Cell Signaling Technology, 9701), rabbit anti-phospho-histone H2A.X (Ser 139) (20E3) (Cell Signaling Technology, 9718), mouse anti-cyclin B1 (Cell Signaling Technology, 4135). Horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology.

Immortalized bronchial epithelial cell line 16HBE cells were obtained from Procell Technology (Wuhan, China). They were cultured in RPMI 1640 (Gibco) at 37°C with 5% CO₂ and cocultured with 10% foetal bovine serum (Biological Industries). According to instruction, we dissolve the LPS powder into different concentrations and stimulate 16HBE for 12 hours.
Antibodies used were rabbit anti-LC3B (Cell Signaling Technology, 3868), rabbit anti-SQSTM1/p62 (Cell Signaling Technology, 8025), and mouse anti-GAPDH (Beyotime, AF5009). Lipopolysaccharide (Sigma-Aldrich, L4391), DAPI (Solarbio, C0065), and RAPA (Sigma-Aldrich, 553210) were used in this study.

Antibodies and their manufacturers were: rat anti-BPLF1 [56 (link)] (WB 1:1600, 2E5 supernatant) was from Helmholtz Zentrum (2E5); mouse anti-FLAG clone M2 (WB 1:4000, IF 1:400; F1804) and rabbit anti-LC3B (WB 1:1000, IF 1:200; L7543) were from Sigma-Aldrich; goat anti-FLAG (1:400; ab1257) and rabbit anti-SQSTM1/p62 (IP 1–2 μg/mg of lysate; ab101266), rabbit anti-GFP (WB 1:2000, ab290) and rabbit IgG isotype control (ab172730) were from Abcam; mouse anti-SQSTM1/p62 (WB 1:1000 IF 1:200; 610,832) was from BD Biosciences; rabbit anti-SQSTM1/p62 (WB 1:1000, 8025S) was from Cell Signaling Technology; mouse anti-HA clone 12 CA5 (WB 1:2000; 11,583,816,001) from Roche; mouse IgG isotype control (14–4732-82) was from Invitrogen; Alexa Fluor 488-, 555-, 594- and 647-conjugated secondary antibodies were from Thermo Fisher (A21206, A31570, A11032 and A21447, respectively).