Figure 5.

Sensitivity of cell surface CD47 to Cytochalasin D is unchanged in protein 4.2 deficient erythroblasts compared to control erythroblasts. (A) To assess the efficiency of the lentiviral pLKO.1 protein 4.2 shRNAs 1-4, transduced cells were differentiated until the polychromatic (T72) erythroblast stage, lysed and separated on SDS-PAGE (0.5 × 10⁶ cells/lane) against a Scramble control shRNA. (B) Erythroblasts, transduced with protein 4.2 shRNA 1 or the Scramble control shRNA, were taken every 24 hours during terminal differentiation, lysed and separated on SDS-PAGE (10⁶ erythroblasts/lane) (n = 4). Western blots were then probed with a rabbit anti-C terminal band 3 (YNTU), BRIC273 (anti-protein 4.2) and anti-GAPDH as a loading control. (C) Protein 4.2 deficient erythroblasts and scramble shRNA control erythroblasts, at T0 and T48, were incubated for 30 minutes with Cyt D (5 µM), or a DMSO control. Erythroblasts were then labelled with BRIC32 followed by APC-conjugated monoclonal IgG1 anti-mouse secondary antibody. Surface levels of CD47 were assessed by flow cytometry (Average MFI as a % of the DMSO control (n = 3); **p ≤ 0.01, *p ≤ 0.05 using the Students T test). 10,000 events were detected using a MACSQuant Analyser 10 flow cytometer and data was analysed using FlowJo version 10.