Horseradish peroxidase conjugated goat anti mouse

ADPKD cells were seeded onto T25 culture flasks at a density of 2.5 × 10⁵ cells/flask. The cells were incubated with test agents depending on the experiments. After treatment, cells were lysed with Triton X-100 lysis buffer containing protease inhibitors. The supernatant from whole-cell lysates was collected by centrifugation. For p53 activation, nuclear and cytoplasmic proteins were separated using the NucBuster Protein Extraction Kit (Novagen, Rockland, USA). Protein concentrations were determined using the Bradford assay. Later, 30 μg total protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated protein bands were blotted onto polyvinylidene fluoride membranes (Bio-Rad). The membranes were blocked with 5% bovine serum albumin and then probed with primary antibodies for p-MEK-1/2, p-ERK-1/2, ERK-1/2, p53, caspase-9, caspase-8, caspase-3, or β-actin. The protein of interest was detected by incubation with secondary horseradish peroxidase-conjugated goat anti-mouse (Santa Cruz Biotechnology) or donkey anti-goat antibodies (Santa Cruz Biotechnology) and visualized with an enhanced chemiluminescence solution from GE Healthy Care Life Science using a ChemiDoc MP system (Bio-Rad). β-Actin was used as a control.

Cytosolic protein was isolated from cells in lysis buffer A, containing Hepes (10 mM), KCl (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (1 mM), PMSF (0.5 mM), Protease Inhibitor Cocktail (15 µL/mL; Sigma-Aldrich), Igepal CA-630 (0.5%; Sigma-Aldrich), pH 7.9. The suspension was centrifuged, and the supernatant (cytosolic extract) was stored. Nuclear extracts were obtained by incubating the pellet obtained from the cytosolic extract in lysis buffer B, containing HEPES (20 mM), NaCl (0.4 M), EDTA (1 mM), EGTA (1 mM), DTT (1 mM), PMSF (0.5 mM), Protease Inhibitor Cocktail (15 µL/mL), pH 7.9. Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). To determine specific protein contents, 20 µg of nuclear cell lysates were analyzed by immunoblotting using antibodies against proliferating cell nuclear antigen (PCNA; 1:100; Santa Cruz Biotechnology INC, Dallas, TX, USA), Cyclin D1 (1:500; Cell Signaling Technology Inc., Danvers, MA, USA) and cleaved Notch 1 (Val1744) (1:200; Cell Signaling Technology) as primary antibodies, and horseradish peroxidase-conjugated goat anti-mouse (1:10000; Santa Cruz Biotechnology) and goat anti-rabbit (1:10000; Santa Cruz Biotechnology) as secondary antibodies. Transcription factor II B (TFIIB; 1:1000; Cell Signaling Technology) was used as loading control.

Total protein was extracted from 50 handpicked Day 1 adult animals grown on OP50 bacteria at 20°C. Animals were washed thoroughly in M9 buffer and centrifuged, and the pellets were lysed in 10 μl of 6x SDS sample buffer. The entire extract was separated by 4–20% SDS-PAGE (Thermo Fisher Scientific, Waltham, MA) and transferred to a PVDF membrane (Millipore, Hayward, CA). Immunoblotting was performed using primary anti-GFP (diluted 1:1000; Santa Cruz Biotechnology, Dallas, TX), anti-mCherry (diluted 1:500; Clontech, Mountain View, CA), and anti-LGG-1 (diluted 1:1000; sample kindly obtained from Abgent, San Diego, CA) antibodies and secondary horseradish peroxidase-conjugated goat anti-mouse (diluted 1:2000; Santa Cruz Biotechnology, Dallas, TX) or goat anti-rabbit (1:2000; Cell Signaling Technology, Danvers, MA) secondary antibodies. Immunoblots were developed using enhanced chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA).

NLRP3 (AG-20B-0014, AdipoGen, CA, USA); IL1β (ab9722, Abcam, MA, USA); Anti-CD63 antibody (ab216130, Abcam, MA, USA); Anti-CD9 antibody (ab92726, Abcam, MA, USA), Anti-TSG101 antibody (ab125011, Abcam, MA, USA), Anti-Alix antibody (ab275377, Abcam, MA, USA), Anti-Calnexin antibody (ab133615, Abcam, MA, USA); Anti-PSD95 antibody (ab2723, Abcam, MA, USA); Anti-GAD65 antibody (ab239372, Abcam, MA, USA); Anti-Gephyrin antibody (ab181382, Abcam, MA, USA); Anti-vGLUT1 antibody (AB5905, Millipore, Burlington, MA, USA), GFP expressing plasmid (13031, Adgene, Watertown, MA, USA); β-actin (A5316, Sigma- Aldrich, MO, USA); horseradish peroxidase conjugated goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, TX, USA) and horseradish peroxidase conjugated goat anti-mouse (sc-2005, Santa Cruz Biotechnology, TX, USA); human primary microglia (Cat # 1900; Sciencell research laboratory, CA, USA) and BV2 microglial cell line was received from Dr. Sanjay Maggirwar (University of Rochester Medical Center, Rochester, NY, USA).

Six days after lentiviral infection, SMMC-7721 cells were collected, washed with ice-cold PBS and lysed in 2× SDS sample buffer (100 mM Tris-Hcl (pH 6.8), 10 mM EDTA, 4% SDS, 10% Glycine). Equal amount of protein samples (30 μg) were run on the 10% SDS-PAGE gel at 50 V for 3 h. The proteins were then transferred to polyvinylidene fluoride (PVDF) membrane at 300 mA for 1.5 h. The membrane was blocked by 1% bovine serum albumin (BSA) in TBST at RT for 1 h and incubated with primary antibodies, mouse anti-USP39 (1:100, Sigma Aldrich, SAB1407042) , rabbit anti-PARP (1: 1000, Cell Signaling Technology, #9542), rabbit anti-Caspase 3 (1: 500, Cell Signaling Technology, #9661), rabbit anti- Caspase 9 (1:1000, Proteintech Group Inc., 10380-1-AP), rabbit anti-Bax (1: 500, Cell Signaling Technology, #2774) and rabbit anti-GAPDH (1:100,000, Proteintech Group Inc., 10494-1-AP) overnight at 4°C. After washed by TBST, membranes were incubated with horseradish peroxidase conjugated goat anti-mouse (1:5,000, Santa Cruz, SC-2005) and goat anti-rabbit (1:5,000, Santa Cruz, SC-2054) secondary antibodies for 2 h at room temperature. The membrane was washed by TBST and signals were detected by enhanced chemiluminescence kit (Pierce).

Lentivirus-infected HCT116 cells were collected, washed with ice-cold PBS and lysed in 2 × SDS sample buffer (100 mM Tris-HCl (pH 6.8), 10 mM EDTA, 4% SDS, 10% glycine). Samples containing equal amounts of protein (30 μg) were run on a 10% SDS-PAGE gel at 50 V for 3 h. Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane at 300 mA for 1.5 h. Membranes were blocked using 1% BSA in TBST at RT for 1 h and then incubated with the primary antibodies anti-HOXC6 (1:100, Novus, NB100-92309), autophagy markers LC3A/B, Atg5, and Atg7 (1: 1000, Cell Signaling Technology, Autophagy Antibody Sampler Kit #4445), anti-SQSTM1/p62 (1:500, Cell Signaling Technology, #8025) or anti-mTOR and p-mTOR (1:1000, Cell Signaling Technology, #9964S) overnight at 4°C. After washing in TBST, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse (1:5,000, Santa Cruz, SC-2005) and goat anti-rabbit (1:5,000, Santa Cruz, SC-2054) secondary antibodies for 1 h at RT. Membranes were washed in TBS-T and signals were detected using an enhanced chemiluminescence kit (Pierce, USA).