Anti glyceraldehyde 3 phosphate dehydrogenase

Monoclonal antibodies used were BRIC32, BRIC126 and fluorescein isothiocyanate (FITC)-BRIC126 (CD47), BRIC222 (CD44) BRIC273 (protein 4.2), BRIC71 (band 3; IBGRL, Bristol, UK), P5D2 (β1 integrin; Developmental Studies Hybridoma Bank, University of Iowa), PE conjugated transferrin receptor (CD71; BD Biosciences), CD71 (sc7327), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma-Aldrich), a C-terminal band 3 antibody (YNTU^{24 (link)}) and a previously reported anti-lysosomal associated membrane protein 1 (LAMP1 270c^{43 (link)}). The unpublished rabbit polyclonal anti-CD47 isoform 3 and 4 specific antibody and the previously described polyclonal antibody recognising the extracellular domain to CD47 (CD47out1^{20 (link)}) were made by Dr. William Mawby, University of Bristol.

Cells were lysed in ice cold RIPA buffer, incubated for 30 min in 4 °C and centrifuged at 14,400× g for 20 min at 4 °C. Samples were resolved using SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (PVDF) (Thermo Fisher Scientific). USP8 protein was detected with USP8 Rabbit polyclonal antibody (27791-1-AP, Proteintech, Rosemont, IL, USA), p27 protein was detected with Anti-p27 KIP1 antibody (ab32034, Abcam, Waltham, MA, USA). Reference proteins Lamin A/C and Glyceraldehyde-3-Phosphate Dehydrogenase were detected with Lamin A/C antibody (sc-20681, Santa Cruz Biotechnology, Dallas, TX, USA) and Anti-Glyceraldehyde-3-Phosphate Dehydrogenase (#MAB374, Sigma-Aldrich), respectively. Anti-rabbit antibody conjugated to HRP (#7074, Cell Signalling, Danvers, MA, USA) was used as secondary antibody. SuperSignal West Pico Chemiluminescent Substrate (no. 34087, Thermo Fisher Scientific) and the CCD digital imaging system Alliance Mini HD4 (UVItec Limited, Cambridge, United Kingdom) were used for visualization. Densitometry was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).