Rabbit anti lc3b

Cell extracts were lysed in 10x RIPA solution (Merck Millipore) and or mouse striatal punches were homogenized in a urea/DTT solution, both containing a cocktail of protease inhibitors (Roche pharmaceuticals), followed ultrasound sonication. Protein concentration levels were determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific) for cell lysates and using NZYBradford reagent (Nzytech) for mouse samples. Sixty to eighty protein µg were resolved in SDS-polyacrylamide gels (7.5% and 12%). Followed by protein transfer to PVDF membrane (Merck Millipore) and antibody probing using the following antibodies: mouse anti-ataxin-2 (1:1000, ref. 611378, BD Biosciences); mouse anti-IC2 (polyQ chain) (1:1000, ref. MAB1574, Merck Millipore); rabbit anti-LC3B (1:1000, ref. NB100-2220, Novus); rabbit anti-SQSTM1 (1:1000, ref. 5114S, Cell Signaling); mouse anti-β-actin (1:5000, ref. A5316, Sigma Aldrich); rabbit anti-DARP-32 (1:1000, ref. AB10518, Merck Millipore). Optical densiometric analysis was carried out using Image J software (USA).

Renal tissue and cells were harvested and lysed by RIPA lysis buffer (Wuhan Goodbio Technology, China) containing cocktail protease inhibitors (Wuhan Goodbio Technology, China) for 30 min on ice. The total protein was obtained by high-speed centrifugation at low temperatures. Protein concentrations were quantified by a BCA protein assay kit (Beyotime Institute of Biotechnology, China), and 30 µg protein was loaded, separated on 10% or 12% SDS-PAGE, and transferred to nitrocellulose membranes. Then membranes were blotted at 4 °C overnight with mouse anti-VEGF-C (Santa Cruz, USA; 1:250), rabbit anti-Prox-1 (Angiobio, USA; 1:1000), rabbit anti-LYVE-1 (Novus, USA; 1:1000), Hamster anti-Podoplanin (Angiobio, USA; 1:200), rabbit anti-Collagen1 (Novus, USA; 1:2000), rabbit anti-α-SMA (Abcam, USA; 1:4000), rabbit anti-PDGFR-β (Abcam, USA; 1:2000), mouse anti-iNOS (Santa Cruz, USA; 1:200), rabbit anti-Arginase (Santa Cruz, USA; 1:400), rabbit anti-LC3B (Sigma-Aldrich, USA; 1:1000), rabbit anti-p62 (Abcam, USA; 1:5000), mouse anti-GAPDH (Wuhan Goodbio Technology, China; 1:2000), then were incubated with HRP-conjugated anti-IgG (Jackson ImmunoResearch, USA; 1:4000), finally were detected by ECL (Pierce, USA). Image capture and analysis were conducted by Bio-RAD (USA).

Calcium oxalate (Sigma, 455997), 3-methyladenine (Sigma, M9281), rapamycin (Sigma, R0395), 4′,6-diamidino-2-phenylindole (Sigma, D8417), N-acetyl-L-cysteine (Sigma, A7250), catalase (Millipore, 219261-100KU), 2′,7′-dichlorofluorescin diacetate (Sigma, D6883), Lipofectamine 3000 (Invitrogen, L3000008), and rabbit anti-LC3B (Sigma, L7543) for western blot (WB) (1:2000), mouse anti-BECN1 (Cell Signaling Technology, 3495) for WB (1:1000). rabbit anti-LC3B (Abcam, ab51520) (1:1000) and mouse anti-BECN1 (Abcam, ab114071) were used for immunohistochemistry (IHC) (1:500). Mouse monoclonal anti-GAPDH (Proteintech, 60004-1-Ig) was used for WB (1:5000). Mouse and rabbit HRP-conjugated antibodies were obtained from Zhongshan Golden Bridge Biotechnology (ZB-2305, ZB- 2301).

CDDP and rapamycin were purchased from Sigma‐Aldrich (St. Louis, MO, USA). A 1 mg/ml stock solution of CDDP was made in PBS, stored at −20°C and diluted in fresh medium to various concentrations prior to use. rapamycin was dissolved in dimethyl sulphoxide (DMSO) in a 1 μM stock solution and stored at −20°C. Foetal bovine serum (FBS), antibiotic mixtures, trypsin and PBS were purchased from Life Technologies (Carlsbad, CA, USA). Rabbit anti‐p‐Akt, Akt, p‐p70S6K, p70S6K, p62, cleaved caspase 3, GAPDH and mouse anti‐PPP2R2A were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti‐LC3B was purchased from Sigma‐Aldrich. LY294002 was purchased from Cell Signaling Technology. T24 and 5637 cell lines were cultured in RPMI‐1640 medium (Life Technologies) containing 10% FBS, 100 units/ml penicillin and 0.1 mg/ml streptomycin. Cells were maintained in an atmosphere of 5% CO₂ at 37°C.

For Western blot (WB) and immnufluorescence, we employed: rabbit anti-LRP1β (Sigma–Aldrich Canada), rabbit anti-LC3B (Sigma–Aldrich), mouse anti-β-actin (GenScript), mouse anti-GM130 (Sigma), anti-rabbit HRP (Sigma), mouse anti-HRP (Sigma), Alexa Flour 488 anti-rabbit (Sigma), Cy3 anti-rabbit (Sigma), Cy3 anti-mouse, and Alexa Flour 594 anti-rabbit (Sigma).