Western Blot Analysis of Cellular Proteins

Total protein was collected from cells with RIPA lysis Mix. Western blotting assay was performed as previously described [23 (link)]. Briefly, 60 μg protein extractions were loaded via SDS-PAGE and transferred onto nitrocellulose membranes (absin, China), then incubated with primary antibodies for 2 hrs at temperature, then plated at 4 °C overnight, the membranes were incubated in 5% non-fat milk blocking buffer for horizontal mode 3 h. After incubation with secondary antibodies IRDye700/800 Mouse or Rabbit (Lincoln, Nebraska, USA), the membranes were scanned using an Odyssey, and data were analyzed with Odyssey software (LI-COR, USA). Primary antibodies list: DDR1 (SAB1300850, Sigma, USA), Vimentin (10366-1-AP, Proteintech, USA), N-cadherin (22018-1-AP, Proteintech, USA), E-cadherin (20874-1-AP, Proteintech, USA), GLUD1 (14299-1-AP, Proteintech, USA), GLS1 (12855-1-AP, Proteintech, USA), SLC1A5 (20350-1-AP, Proteintech, USA), STAT3 (10253-2-AP, Proteintech, USA), p-STAT3(705) (ab76315, Abcam, UK), p-STAT3(727) (ab32143, Abcam, UK), GAPDH (60004-1-Ig, Proteintech, USA).