Anti shmt2

Fifty micrograms of total protein from each sample was separated by 12% SDS/PAGE and electro-transferred onto PVDF membranes (Millipore, Boston, MA) for immunoblotting analysis. The primary antibodies including Anti-IGF2 (1:500, Abcam), Anti-AKT2 (1:500, Abcam), Anti-SHMT2 (1:300, Abcam), and Anti-β-actin (1:800, Abcam) were used to incubated with the membranes at 4°C overnight. After incubation with appropriate Horseradish Peroxidase-conjugated secondary antibodies, the blots were detected using Immobilon ECL Kit (Millipore) in a ChemiDoc XRS Imaging System and analyzed by Quantity One software (Bio-Rad).

The transfected J82 and T24 cells were lysed using protein inhibitor modified RIPA lysate (Thermo Fisher) on ice, and the total cell protein was extracted. After accurate loading, electrophoresis separation was performed on a 10% SDS-PAGE gel, and then transferred to a superior PVDF membrane. The PVDF membrane was blocked with skim milk (BD) for 2 h. The following primary antibodies were added and incubated overnight at 4 °C, anti-SHMT2(1:1,000, ab224428; Abcam, Cambridge, UK), anti-Tubulin(1:10,000, ab176560; Abcam, Cambridge, UK), anti-MMP7(1:1,000, ab205525; Abcam, Cambridge, UK), anti-Integrin β-4(1:1,000, ab182120; Abcam, Cambridge, UK), anti-Laminin β-4(1:500, Sc-130540; Santa Cruz, CA, USA), anti-SHMT1(1:800, Cat.14149–1-AP; Proteintech, Wuhan, China), anti-E-cadherin (1:2,000, Cat.60335–1-Ig; Proteintech, Wuhan, China), anti-N-cadherin (1:2,000, Cat.66219–1-Ig; Proteintech, Wuhan, China), anti-MMP2 (1:2,000, Cat.66366–1-Ig; Proteintech, Wuhan, China), anti-GAPDH (1:10,000, Cat.60004–1-Ig; Proteintech, Wuhan, China). The membrane was washed the next day and secondary antibodies were added and incubated for 30 min. After washing, the membrane was treated with ECL chemiluminescence reagent for 1 min and finally imaged using a fluorescence imaging system. The gray value of protein bands was analyzed using Image J software and the experimental data were recorded.