G box chemi fluorescent chemiluminescent imaging system

Cells were washed with ice-cold DPBS and lysed on ice using the following buffer: 20 mM Tris, HCl, 100 mM NaCl, 1% Triton X-100 at PH 7.4 containing protease inhibitors (cOmplete ULTRA Cat#05892970001 Roche). The proteins were separated on SDS–PAGE and electrotransferred onto nitrocellulose membrane. After transfer, the membrane was saturated in DPBS containing 0.1% Tween 20 and 5% milk. Primary antibodies were added overnight at 4 °C or for 2 h at room temperature depending on the antibody. The membranes were washed with DPBS and incubated for 1 h at room temperature with appropriate secondary antibody coupled with peroxidase. ECL plus kit (Cat#32132 Thermo Scientific) was used for protein detection. Chemiluminescent signal was detected by G:BOX Chemi Fluorescent & Chemiluminescent Imaging System from SYNGENE. Blot quantification was done using ImageJ software.
Blot Scans are presented in the figures and the uncropped scan are supplied in Supplementary Fig. 15.

The cells were washed with ice-cold Dulbecco’s Phosphate Buffered Saline (DPBS) and lysed on ice in the following buffer: 20 mM Tris, HCl, 100 mM NaCl, 1% Triton X100, and 10 mM EDTA at PH 7.4 containing a protease and phosphatase inhibitor cocktail (Complete™ ULTRA Cat#05892970001 Roche). The proteins were separated on SDS (sodium dodecyl sulfate) polyacrylamide gel and electro-transferred onto nitrocellulose membranes. After transfer, the membranes were saturated in DPBS containing 0.1% Tween 20 and 5% milk. Primary antibodies were added overnight at 4 °C or for 2 h at room temperature depending on the antibody. The membranes were then washed with DPBS and incubated for 1 h at room temperature with appropriate secondary antibodies coupled with peroxidase. The ECL plus kit (Cat#32132) and SuperSignal™ West Femto Maximum Sensitivity Substrate (Cat#34095), obtained from Thermo Scientific, were used for protein detection. Chemiluminescent signals were detected by the G: BOX Chemi Fluorescent & Chemiluminescent Imaging System from SYNGENE. The blots were quantified using Image J 1.53t.