Ab224428

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.

After transfection or lapatinib treatment, cells were lysed in RIPA buffer (Beyotime, shanghai, China). The protein levels were determined using immunoblotting analyses following a method described previously [25 (link)] with the following primary antibodies, anti-ERRα (ab76228, Abcam, Cambridge, MA, U.S.A.) and anti-SHMT2 (ab224428, Abcam), and a secondary anti-mouse antibody conjugated with horseradish peroxidase (Jacksons Immunoresearch, Mill Valley, CA, U.S.A.). GAPDH protein levels were detected with anti-GAPDH antibody (ab8245, Abcam) and used as an internal control to normalize the levels of target proteins.