Anti gapdh antibody sc 25778

Foetal bovine serum (FBS) was purchased from PAA Laboratories (GE Healthcare Life Sciences, Little Chalfont, UK) or Labtech International, Ltd. (Heathfield, UK). Precision Plus Protein Colour standards and nitrocellulose were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Human recombinant EGF (PHG0313) was from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Alexa Fluor 555-EGF (E-35350) was from Molecular Probes; Thermo Fisher Scientific, Inc. EGFR (D38B1) XP^® rabbit antibody (Alexa Fluor 488-conjugate; 1:100; cat. no. 5616) and EGFR rabbit antibody (D38B1; 1:100; cat. no. 4267) were from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-early endosome antigen 1 (EEA1) antibody (1G11) Early Endosome Marker (ab70521; 1:1,000) was from Abcam (Cambridge, UK). Anti-GAPDH antibody (sc-25778; 1:1,000) was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). VectaShield^® mounting medium was from Vector Laboratories, Ltd. (Peterborough, UK). All other reagents were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany), unless stated otherwise. Aspirin analogues, detailed in Table I, were synthesised in-house using previously described methods (26 (link),27 (link)).

Total protein was extracted from cells using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was determined using the BCA method. The proteins were separated by the SDS-PAGE using the Mini-PROTEAN Tetra instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA), then transfered onto PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were blocked by 5% BSA and incubated overnight at 4°C with specific primary antibody rabbit polyclonal anti-SOX7 antibody (ab220293, 1:1,000; Abcam, Cambridge, UK). After that, the membrane was incubated in the secondary antibody goat polyclonal anti-rabbit IgG H&L secondary antibody (ab150077, 1:2,000; Abcam) at room temperature for 1 h. Anti-GAPDH antibody (sc-25778; 1:3,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used as a loading control at 4°C overnight. Signal was measured using the GelDoc 2000 instrument (Bio-Rad Laboratories, Inc.).