Anti lc3b l7543

Transfection reagents Lipofectamine 2000 and Lipofectamine 3000 were purchased from Invitrogen, RNA synthesis inhibitor actinomycin D, protease inhibitor MG132, lysosomal inhibitors bafilomycin and hydroxychloroquine, CDK inhibitor RO-3306, and thymidine were purchased from Sigma-Aldrich. λPPase was purchased from New England BioLabs. Anti-CELF6 (ab173282) and anti-GFP (ab1218) were obtained from Abcam, anti-p21^Waf1/Cip1 (#2947), anti-p27^Kip1(#3686), anti-Gadd45α (#4632), anti-Cyclin B1(#12231), anti-β-TrCP (#4394), anti-Wee1 (#13084), anti-cleaved PARP (#5625), anti-cleaved Casp3 (#9661), anti-cyclin E1 (#20808), and anti-Ubiquitin (#3936) antibodies were purchased from Cell Signaling Technology, anti-P53 (10442-1-AP), anti-GFP (66002-1-1 g), anti-His (66005-1-1 g), anti-β-tubulin (60008-1-1 g), and anti-GAPDH (10494-1-AP) were obtained from Proteintech, anti-LC3B (L7543) was obtained from Sigma-Aldrich.

Transfected cells were collected at 48 h post-transfection and reactivated Akata cells were harvested at the indicated time. Cell pellets were lysed in lysis buffer (50 mM Tris·HCl pH 6.8, 2% SDS, 2% β-mercaptoethanol), subjected to SDS-PAGE, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Amersham). The membranes were blocked with 5% bovine serum albumin (BSA) or skim milk powder and incubated at 4 °C overnight with the indicated antibodies. Anti-ZEBRA (sc-53904; 1/5000), anti-β-actin (sc-47778; 1/5000), and anti-HA (sc-805; 1/1000) antibodies were purchased from Santa Cruz; anti-LC3B (L7543) (1/4000) and anti-Sequestosome1 (SQSTM1/p62) (5114T; 1/4000) were obtained from Sigma and Cell Signaling Technology, respectively. Anti-sera against BALF0/1 were prepared by immunizing a rabbit with the recombinant protein of BALF0/1, produced as previously described [37 (link)], and used for immunoblotting analysis at a dilution of 1/500. Horseradish peroxidase-conjugated goat antibodies directed against mouse (Cell Signaling Technology, Leiden, Netherlands) or rabbit (Amersham, Saint-Quentin Fallavier, France) immunoglobulins were used as secondary antibodies (1/10,000). Immunodetection was performed using the ECL detection system according to the manufacturer’s instructions (Amersham).

Whole-cell lysates were resolved by SDS-PAGE and electrophoretically transferred to Hybond PVDF membranes (Amersham, GE Healthcare GmbH, Solingen, Germany). PVDF membranes were treated overnight with primary antibodies at 4 °C: anti-SQSTM1/p62 (#5114), anti-Beclin-1 (D40C5), anti-AMPKα (D5A2) (#5831), anti-cathepsin D (E179) (#69854), anti-phospho-Akt (Ser473) (D9E) (#4060), anti-Akt (pan) (C67E7) (#4691) were from Cell Signaling Technology (Danvers, MA, USA); anti-LC3B (L7543), and anti-BCL2 (BCL2-100) (B3170) were from Sigma–Aldrich (Merck KGaA); anti-human LAMP-1 (611042) (clone 25/Lamp-1) was obtained from BD Biosciences (Franklin Lakes, NJ, USA); anti-phospho-Beclin-1 (Ser295) (#PA5-35394) and anti-PBS TFEB (#PA1-9109) were obtained from Invitrogen (ThermoFisher Scientific Waltham, MA, USA). A mouse monoclonal anti-GAPDH antibody (6C5) (MAB-10578) (Immunological Sciences, Roma, Italy) was used as the loading control. After washing, PVDF membranes were incubated with the appropriate secondary antibody conjugate with horseradish peroxidase at room temperature for 1 h. Detection by chemiluminescence and analysis were performed as previously described [12 (link)].

Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and propidium iodide (PI) were purchased from Sigma. The following antibodies were used for western blot analysis or immunofluorescence: COX IV (4850), NDP52 (60732), and GST (2625) were purchased from Cell Signaling Technology; monoclonal anti-ubiquitin (sc-8017), anti-Tom20 (sc-17764), anti-PINK1 (sc-517353), and anti-Parkin (sc-32282) were purchased from Santa Cruz; anti-LC3B (L7543) was purchased from Sigma; and anti-Tim23 (611222) was purchased from Becton Dickinson and Company. Actin (EM21002) was purchased from HuaBio. GAPDH (ab128915) was purchased from Abcam.