Anti rabbit antibodies

After treatment, cells were washed with PBS and lysed with RIPA buffer with protease inhibitors (Merck, New York) and phosphatase inhibitors (Merck). Equal amounts of protein (40 μg/lane) were resolved by SDS-PAGE, and transferred to PVDF membrane (Roche, Indianapolis, IN). All primary antibodies were incubated overnight at 4°C: anti-Bcl-2 (2870), anti-Bcl-xL (2764), anti-Mcl-1 (5453), anti-Bad (9239), anti-Bid (2002), anti-Bim (2933), anti-Puma (12450), anti-Bak (12105), anti-Bax (5023), anti-ATG5 (8540), anti-ATG7 (2631), anti-Beclin-1 (3738), anti-β-actin (4970), anti-cathepsin D (2284), anti-LC3B (2775), anti-p62 (8025), anti-ubiquitin (3936) (Cell signaling, Beverly, MA), anti-cathepsin B (C6243) (Sigma-Aldrich), and anti-cathepsin L (BMS1032) (eBioscience, San Diego, CA). All primary antibodies were used at a dilution of 1:1000, except for anti-ATG7 protein, which was used at a dilution of 1:500. The membranes were then incubated for 1 h with secondary peroxidase-conjugated antibodies (1:2000) (Anti-rabbit antibodies, Cell Signaling Technology, 7074; Anti-mouse antibodies, Cell Signaling Technology, 7076). Chemiluminescent signals were then developed with Lumiglo reagent (Cell Signaling Technology, 7003) and exposed to X-ray film (FUJIFILM Europe GmbH, Dusseldorf, Germany). The films were analyzed by densitometry with ImageJ software.

Dimethyl sulfoxide (DMSO), 1,4-NQ (98% purity determined by gas chromatography) and anti-GAPDH antibody were from Wako Pure Chemical Industries (Osaka, Japan), Tokyo Chemical Industries (Tokyo, Japan) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Biotin-PEAC₅-maleimide (BPM) and Na₂S₄ were from Dojindo Laboratories (Kumamoto, Japan). Dynabeads M-280-sheep anti-rabbit immunoglobulin G (IgG) was from Invitrogen (Carlsbad, CA, USA). Anti-Akt, anti-CREB, anti-phosphorylated Akt (Ser473), anti-phosphorylated CREB (Ser133), horseradish peroxidase (HRP)-conjugated anti-biotin antibodies, anti-rabbit antibodies and anti-mouse IgG secondary antibodies were from Cell Signaling Technology (Beverly, MA, USA). Escherichia coli BL21 cells and trypsin were from Promega Co. (Madison, WI, USA). Glutathione 4B Sepharose was from GE Healthcare (Chicago, IL, USA). All other reagents were of the highest purity available.

For Western blot analysis, cells were lysed in RIPA buffer (20 mM Tris pH 8.0, 0.1% SDS, 150 mM NaCl, 0.08% sodium deoxycholate, and 1% NP40) supplemented with 1 tablet of protease inhibitor (complete ultra mini-tablet, Roche, Indianapolis, IN, USA) and phosphatase inhibitor (PhosStop tablet, Roche, Indianapolis, IN, USA). A total of 20 µg of total protein was loaded per lane and separated by SDS-PAGE. The separated proteins on the gel were transferred onto the PVDF membrane and were probed for specific antibodies against Rb (cat#9309; Cell Signaling Technology, Danvers, MA, USA), phospho-Rb (cat#8516, Cell Signaling Technology, Danvers, MA, USA), E2F2 (ab209662; Abcam, Cambridge, UK), HK1(cat#2024; Cell Signaling Technology, Danvers, MA, USA) and GAPDH (cat#5174; Cell Signaling Technology, Danvers, MA, USA) at 1:1000 dilution in 5%BSA in 1× TBST overnight at 4 °C. After 4 washes with 1× TBST for 10 min, membranes were incubated with HRP-conjugated anti-mouse (cat#7076; Cell Signaling Technology, Danvers, MA, USA) or anti-rabbit antibodies (cat#7074; Cell Signaling Technology, Danvers, MA, USA) at 1:2000 dilution for 2 h. Images were visualized using the Image Quant LAS 500 system (GE Healthcare Life Sciences, Piscataway, NJ, USA).

Protein from U-87 MG cells was isolated using TRIS lysis buffer (25 mM mercaptoethanol, 1 µl/ml Tween 20, 10 µl/ml protease inhibitor, 50 mM TRIS base, pH=7.5) and sonication for 30 s. 120 µg of denatured protein sample were loaded into each well of the 12% ProSieve 50 (Lonza, ME, USA) modified acrylamide gel for electrophoresis (80 mV for 20 min, then 120 mV for 90 min), followed by transfer onto PVDF membranes (100 mV for 90 min). After blocking (5% skim milk powder in 10 mM TRIS-buffered saline) at 4 °C for 1 h, the blocked membranes were incubated at 4 °C overnight with 1:1000-fold dilutions of primary antibodies against the K_Ca1.1 β subunits or actin: anti-KCNMB1 (nb300-535, rabbit polyclonal, Novus Biologicals, CO, USA), anti-KCNMB2 (MA5-27646, mouse monoclonal, Thermo Fisher Scientific, Waltham, MA, USA), anti-KCNMB3 (ab137041, rabbit monoclonal, Abcam, Cambridge, UK), anti-actin (a2066, rabbit polyclonal, Sigma-Aldrich, MO, USA). After washing three times, blots were incubated 1:10 000-fold diluted secondary anti-mouse (#7076, Cell Signaling Technology, MA, USA) or anti-rabbit antibodies (#7074, Cell Signaling Technology, MA, USA) at 4 °C for 2 h. Blot chemiluminescence was detected using a commercial detection system (Chemidoc XRS, Bio-Rad, Hercules, CA, USA).