Anti lc3b nb100 2220

The immortalized RPTC cell line was originally obtained from Dr. Ulrich Hopfer (Case Western Reserve University) and maintained in DMEM/F-12 medium supplemented with 10% fetal bovine serum and growth factors [47]. The retrovirus packaging cell line (293-Phoenix) was provided by Drs. Xiongjie Jin and Nahid F. Mivechi (Augusta University). The cells were cultured in DMEM medium with 10% fetal bovine serum. Primary antibodies: anti-LC3B (NB100-2220), anti-PINK1 (BC100-494) and anti-FUNDC1 (NBP1-81063) were from Novus Biologicals; anti-SQSTM1 (ab56416), anti-IMMT/MIC60 (ab110329), anti-COX4I1 (ab153709) and anti-PPIB (ab16045) were from Abcam; anti-TOMM20 (sc-11415), anti-PINK1 (sc-517353), anti-PRKN (sc-133167), anti-BNIP3L/NIX (sc-166332) and anti-HSPD1 (sc-13966) were from Santa Cruz Biotechnology; anti-CASP3 (9665) and anti-cleaved CASP3 (9664) were from Cell Signaling Technology; anti DNM1L (BD Biosciences, 611113); anti-ACTB (Sigma-Aldrich, A5316). Secondary antibodies for immunoblot analysis were from Jackson ImmunoResearch Laboratories. CCCP (C2759), chloroquine (C6628) and 3-methyladenine (M9281) were from Sigma-Aldrich. BECN1 peptide (Tat-BECN1, sequence: YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT) and its control peptide (Tat-scrambled, sequence: YGRKKRRQRRRGGVGNDFFINHETTGFATEW) were synthesized by GenScript.

Renal cortical and outer medulla tissues were lysed using 2% SDS buffer with 1% protease inhibitor cocktail (P8340, Sigma-Aldrich). Protein concentration was determined using a Pierce BCA protein assay kit (no. 23225) from Thermo Scientific. Equal amounts of proteins were separated by SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% fat-free milk or 5% BSA for 1 h and subsequently incubated with primary antibodies at 4°C overnight and secondary antibodies for 1 h at room temperature. Primary antibodies used in present study were from following sources: anti-Vimentin 5,741) and anti-cleaved Caspase-3 (Asp175) 9,661) from Cell Signaling Technology; anti-LC3B (NB100-2220) from Novus Biologicals; anti-α-Smooth Muscle Actin (ab5694) and anti-Fibronectin (ab2413) from Abcam; anti-GAPDH (10494-1-AP) from Proteintech; anti-Collagen 1 (AF7001) from Affinity; all secondary antibodies for immunoblot analysis from Thermo Fisher Scientific. Antigen-antibody complexes on the membranes were detected with an enhanced chemiluminescence kit from Thermo Scientific. For quantification, protein bands were analyzed with ImageJ software.

Primary antibodies to β-actin (SC-47778, 1:1000), CaMKK-ß (SC-50341, 1:1000), and AMPKα1/2 (SC-25792, 1:1000) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies to p-CaMKKβ (12818S, 1:500), mTOR (2983S, 1:1000), S6 ribosomal protein (2217S, 1:1000), and p-S6 ribosomal protein (2211S, 1:1000) were purchased from Cell Signaling (Danvers, MA). Anti-p-AMPKα1/2 (11183, 1:1000) and anti-p16 (41296, 1:1000) antibodies were from Signalway (College Park, MD, USA). Anti-LC3B (NB100-2220, 1:500) and anti-p21 (NBP2-29463, 1:500) antibodies were purchased from Novus (St. Louis, MO, USA). Anti-p-mTOR (ab63552) and anti-Ki-67 (ab15580) antibodies were purchased from Abcam (Cambridge, UK). Anti-mouse (31430), anti-rabbit (31460), anti-goat (31402), and Alexa Fluor 488 (A11008) immunoglobin G secondary antibodies (1:5000) were purchased from Invitrogen (Carlsbad, CA, USA). The anti-OR2H2 primary antibody (1:1000) was purchased from Antikoerper-online.de (Aachen, Germany). L-Cis diltiazem was from Abcam (Cambridge, UK). SQ22536 and thapsigargin were purchased from Enzo Life Science (Farmingdale, NY, USA) and U73122 was from Sigma-Aldrich (St. Louis, MO, USA). Aldehyde 13-13 was obtained from Henkel (Düsseldorf, Germany). A769662, an AMPK agonist, was purchased from Cayman (Ann Arbor, MI, USA). Rapamycin was obtained from LC Laboratories (Woburn, MA, USA).

Anti-SQSTM1 (5114), anti-COXIV (11967), and anti-activated caspase-3 (9664) were from Cell Signaling Technologies; anti-LC3B (NB100-2220) were from Novus Biologicals; anti-TIMM23 (sc-514463) and anti-TOMM20 (sc-11415) were purchased from Santa Cruz Biotechnology; anti-KIM1/HAVCR1 (hepatitis A virus cellular receptor 1) from R&D Systems; anti-cyclophilin B, anti-macrophage (RM0029-11H3), anti-neutrophil (NIMP-R14), and anti-BNIP3 (ab109362, ab10433) were obtained from Abcam; anti-β-actin (A5316) was from Sigma-Aldrich. All secondary antibodies, DHE (D11347) and collagen I (A1048301), were purchased from Thermo Fisher Scientific, and TUNEL assay kit (12156792910) was obtained from Roche Life Science.

Glioblastoma cell lines LN229 and T98G were treated and collected after 24 h in lysis buffer (50 mM HEPES, pH 7.5, 10% glycerol, 0.1% Triton X-100, 1 mM dithiothreitol, 150 mM NaCl, 2 mM MgCl₂, and protease inhibitor cocktail). Then, 30 μg of protein was taken out from the cell lysate as the input sample. Anti-ALDH1a3 (ab129815, abcam, Berlin, Germany) and anti-IgG (7074p2, CST, Leiden, Netherland) were added to the lysate for immunoprecipitation (containing 30 μg of protein), and then incubated at 4 °C overnight. Next, 2 mg of protein-G-Sepharose beads were added to each immunoprecipitation sample for 3 h in 4 °C. After washing, Sepharose beads were collected and diluted with SDS sample buffer (1 M Tris-HCl pH 6.8, SDS, 0.1% Bromophenol Blue, glycerol, and 14.3 M β-mercaptoethanol), and then heated at 100 °C in 5 min. Input samples and immunoprecipitation samples were fractionated by 10% SDS-page gels and read out by Western blotting with anti-LC3b (NB100-2220, Novus bio, Munich, Germany).