Hrp conjugated goat anti mouse or goat anti rabbit secondary antibodies

Protein extracts were prepared in lysis buffer [20 mm Hepes–KOH, pH 7.9, 0.42 m KCL, 25% glycerol, 0.1 mm EDTA, 5 mm MgCl2, 0.2% NP40, 1 mm DTT, 500 μm PMSF, and 1:100 protease inhibitor cocktail (Sigma–Aldrich)]. 50-μg protein samples were run on 12% SDS-PAGE gels, and proteins were transferred to PVDF membranes (Pall Life Sciences, Pensacola, FL, USA) using a Bio-Rad mini Trans-Blot cell at 350 mA for 90 min. After transfer, membranes were blocked in 5% nonfat dry milk in 1 × TBST (150 mm NaCl, 10 mm Tris-HCl, pH 8.0, 0.05% Tween-20) at room temperature for 1 h and then incubated with primary antibody at 4 °C overnight with gentle agitation. Membranes were washed three times in 1× TBST for 10 min, and subsequently incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Pierce Biotechnology, Rockford, IL, USA) at room temperature for 1 h with shaking. Membranes were washed three times in 1 × TBST for 10 min. Proteins were detected with a SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology), and signals were exposed to Hyblot CL Autoradiography films (Denville, Metuchen, NJ, USA). Autoradiography films were scanned, and band signal intensities were measured by densitometry using the ImageJ (NIH, Bethesda, MD) software (version 1.45).

Protein extracts were prepared in RIPA lysis buffer (20 mM Hepes‐KOH, pH7.9, 0.42 M KCL, 25% glycerol, 0.1 mM EDTA, 5 mM MgCl2, 0.2% NP40, 1 mM DTT, 500 μM PMSF, and 1:100 protease inhibitor cocktail (Sigma‐Aldrich, St Louis, MO). 30 µg protein samples were run on 12% SDS‐PAGE gels and proteins were transferred to PVDF membranes (Pall Life Sciences; Pensacola, FL) using a Bio‐Rad mini Trans‐Blot Cell at 350 mA for 90 min. After transfer, membranes were blocked in 5% nonfat dry milk in 1× TBST (150 mM NaCl, 10 mM Tris‐HCl, pH 8.0, 0.05% Tween 20) at room temperature for 1 hr, then incubated with primary antibody at 4°C overnight with gentle agitation. Membranes were washed three times in 1× TBST for 10 min and subsequently incubated with HRP‐conjugated goat anti‐mouse or goat anti‐rabbit secondary antibodies (Pierce Biotechnology; Rockford, IL) at room temperature for 1 hr with shaking. Membranes were washed three times in 1× TBST for 10 min. Proteins were detected with a SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology; Rockford, IL) and signals were exposed to Hyblot CL Autoradiography films (Denville; Metuchen, NJ). Autoradiography films were scanned and band signal intensities were measured by densitometry using the Image J software (version 1.45). γ‐tubulin or ponceau S stain was used as a loading control as indicated.

SDS-PAGE and immunoblotting were performed with standard protocols described previously using 5 to 10% acrylamide gels [57 (link)]. SDS-PAGE gels were stained with CBB (Coomassie brilliant blue) or silver. For immunoblotting, samples separated by SDS-PAGE were transferred to a nitrocellulose or PVDF membrane, stained with CBB or Ponceau S if necessary, incubated with specific primary antibodies and subsequently HRP (horseradish peroxidase)-conjugated secondary antibodies. Immuno-reaction was detected using a TMB (3,3′,5,5′-tetramethylbenzidine peroxidase) substrate kit (Vector Laboratories), Pierce ECL immnoblotting substrate (Thermo Scientific) or ECL prime immunoblotting detection reagent (GE Healthcare). Primary antibodies used were as follows: anti-DYX1C1 CT299 (Rabbit: this study), anti-PF13/KTU (Rabbit: [17 (link)]), anti-IDA10/MOT48 (Rabbit: this study), anti-IC138 (Rabbit: [62 (link)]), anti-IC2 (Mouse: [63 (link)]), anti-Actin (Rabbit: [64 (link)]), anti-p28 (Rabbit: [59 (link)]), and anti-HA 3F10 (Rat: Roche Applied Science) or anti-HA Y11 (Rabbit: Santa Cruz). HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies were commercially purchased from Invitrogen. Anti-PF13/KTU antibody was a generous gift from Dr. David R. Mitchell (SUNY Upstate Medical University).