Protein g ip kit

IP was performed using Protein G IP Kit (Roche, Switzerland) according to the manufacturer’s instruction. For western blotting, cell pellets were solubilized with RIPA buffer (Beyotime Institute of Biotechnology, China) with addition of cOmplete Protease Inhibitors (Roche, Switzerland) and PhosSTOP Phosphastase Inhibitors (Roche, Switzerland), electrophoresed, and blotted onto PVDF membranes. The membranes were incubated with indicated primary antibodies followed by the incubation with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, UK). Protein concentration was calculated by the BCA Protein assay kit (Thermo Scientific Pierce, UK). Blotted proteins were visualized using an enhanced chemiluminescence detection kit (Tiangen Biotech, China). The intensity of the bands was analyzed by Quantity One V 4.62 Software. Antibodies against GNB2L1 (Abcam, USA) was used for both IP and WB, and antibodies against OGT, OGA, GAPDH, E-cadherin, N-cadherin, O-GlcNAc (Abcam, USA) were used in WB.

Protein G IP Kit (Roche, Switzerland) was used for IP according to the manufacturer's instructions. For western blotting, cell pellets were lysed with RIPA buffer (Beyotime Institute of Biotechnology, China) containing complete Protease Inhibitors (Roche, Switzerland) and PhosSTOP Phosphastase Inhibitors (Roche, Switzerland), electrophoresed, then blotted onto PVDF membranes. The membranes were incubated with the indicated primary antibodies, followed by incubation with HRP‐conjugated secondary antibodies (Jackson ImmunoResearch, UK). The protein concentration was calculated using the BCA Protein assay kit (Thermo Scientific Pierce, UK). Blotted proteins were visualized using an enhanced chemiluminescence detection kit (Tiangen Biotech, China), and the intensity of the bands was analyzed using Quantity One V 4.62 Software (Bio‐Rad, Life Science, California, USA).