Ab109012

After incubation with various concentrations of PIONs@ E6 for 24 hours, U251 cells were homogenized in lysis buffer, separated by 10% SDS-PAGE, and transferred to PVDF membranes (IPVH00010, EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% skimmed milk and then incubated with diluted primary antibodies including rabbit anti-P62 (1:10000; ab109012, Abcam, MA, USA), anti-beclin-1(1:2000; ab207612, Abcam), anti-LC3II (1:2000; ab192890, Abcam), anti-LC3I (1:2000; ab192890, Abcam), and anti-GAPDH (1:1000; ab181602, Abcam). Blots were washed with TBS/TWEEN and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (1:5000; KGAA35, Keygen Inc., Nanjing, P.R.C) for 2 hours. After washing with TBS/TWEEN, the blots were developed with the chemiluminescence method (ECL Luminata Crescendo, WBLUR0500, EMD Millipore).

Lysosome inhibitor E64d and proteasome inhibitor lactacystin were purchased from Sigma (E8640, and L6785). 1-(2-aminoethyl) piperazine (AP), trifluoromethanesulfonamide (TFMS), acetazolamide (AZA), 4-phenylbutyric acid (PBA), and methyl-β-cyclodextrin (MβCD) were obtained from Sigma (A55029, 638455, A6011, P21005, and C4555, respectively). CHRN antibody (mAb210, against α1/α3/α5 subunits) and SQSTM1 were obtained from Abcam (ab24719 [no longer available] and ab109012). Antibodies against CAR2 and CAR3 were purchased from Santa Cruz Biotechnology (sc-133111, and sc-50715). Antibodies for ACTB, MAP1LC3A/B, BECN1, ATG5, and ATG7 were obtained from Cell Signaling Technology (3700S, 12741, 3738, 12994, and 8558, respectively). Antibody for FLNC was obtained from Biorbyt (orb326498). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (115–035-003, and 111–035-003). Biotinylation kit and Alexa Fluor 488 labeling kit for CHRN antibody were obtained from Thermo Fisher Scientific (90407, and A30006). Streptavidin-HRP and Tunicamycin were purchased from Sigma (18–152, and T7765). Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide was obtained from Invitrogen (V13245).

Proteins were extracted from OS tissues or cells using Tissue Extracts & Cell Lysates (Santa Cruz, San Diego, CA, USA), and 50 µg proteins were applied to carry out the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 90 min. After proteins were transferred onto the UltraCruz Nitrocellulose Pure Transfer Membranes (Santa Cruz), TBS Blotto A (Santa Cruz) was exploited as a blocking reagent to prevent the binding of no-specific protein-binding signals. Then the membranes were incubated with primary antibodies in the diluted solution at 4°C overnight, followed by the combination of secondary antibody and primary antibodies at room temperature for 1 h to form the protein complex. The objective protein levels were analyzed by detecting the intensity of the immunoconjugated signals through Western Blotting Luminol Reagent (Santa Cruz) under the ImageLab software version 4.1 (Bio-Rad, Hercules, CA, USA). All the antibodies were bought from Abcam (Cambridge, UK): anti-Beclin-1 (ab62557, 1:1000), anti-light chain 3B (anti-LC3B; ab51520, 1:1000), anti-P62 (ab109012, 1:1000), anti-HDAC4 (ab12172, 1:1000), internal control anti-GAPDH (ab9485, 1:3000) and secondary antibody goat anti-rabbit IgG/HRP (ab205718, 1:5000).