Anti halotag g9211

Regular immunoblotting was performed for protein detection following SDS‐PAGE electrophoresis. The primary antibodies used for western blot analysis include: anti‐FLAG^® (F7425) (1:2000), anti‐FLAG^® M2 (F1804) (1:4000), anti‐c‐Myc (C3956) (1:2000) from Sigma‐Aldrich (St. Louis, MO); anti‐Myc Tag, clone 4A6 (05‐724) (1:4000) from Millipore Corporation (Billerica, MA); anti‐ CK1α1 [EPR1961(Mendoza et al. 2007)] (ab108296) (1:2000), anti‐CK1δ [AF12G4] (ab85320) (1:4000), anti‐CK1ε [AF6C1] (ab82426) (1:2000) from abcam^® (Cambridge, MA); anti‐HaloTag^® (G9211) (1:1000) from Promega Corporation (Madison, WI). Two secondary antibodies were used: ECL anti‐rabbit IgG (NA934V; GE Healthcare; Little Chalfont, U.K.) and anti‐mouse IgG+IgM HRP (ab47827; abcam^®; Cambridge, MA).

Cells were harvested following transfection or treatment at specific time points in lysis buffer (0.128 M β-mercaptoethanol, 40 mM tris, 10% glycerol, 1% SDS and 0.01% bromophenol blue) containing PhosSTOP phosphatase and Complete protease inhibitor cocktails (both from Roche). Proteins were separated by SDS-PAGE on a 10% polyacrylamide gel, transferred to nitrocellulose membrane and probed with the primary antibody at 4 °C overnight. Antibodies for PERP (ab5986), SERCA2b isoform specific (ab137020) and GAPDH (ab8245) were purchased from Abcam, and antibodies for LC3B (#3868) and p62 (#8025) were from Cell Signalling Technology (Leiden, The Netherlands). Anti-HaloTag (G9211) was from Promega and anti-p53 (P6874) was from Sigma-Aldrich. Immunocomplexes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody and detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) or Radiance Plus Chemiluminescent Substrate (Cambridge Bioscience, Cambridge, UK) and a Bio-Rad Chemidoc Imaging System. Membranes were incubated in stripping solution at 55 °C for 25 min and sequentially re-probed.