Figure 5
From: Heat shock protein A2 is a novel extracellular vesicle-associated protein
Specificity of HSPA2 detection in SEC-purified EVs derived from HSPA2-knockout and HSPA2-overproducing NCI-H1299 cells. (a) Analysis of HSPAs in HSPA2-knockout cells modified by CRISPR/Cas9 system. WT, wild-type cells, CTR I, CTR II, modified control cells; MIX I, MIX II; pools of isogenic clones with partial and full knockout of the HSPA2 gene expression, respectively. (b) Levels of HSPA2; positive (CD63, CD81, TSG101) and negative (GM130) protein markers in EVs produced by wild-type (WT), modified control (CTR I, CTR II) and HSPA2-knockout cells (MIX I, MIX II). (c) Expression of HSPAs and GFP-HSPA2 fusion protein in WT, control GFP tag-overexpressing (p-GFP); GFP-HSPA2-overexpressing (p-GFP-A2) cells. Stable cell lines were established by lentiviral transduction. (d) Detection of HSPA2 in EVs-enriched SEC fraction derived from WT, control p-GFP and p-GFP-A2 cells. WT HSPA2, 70 kDa; GFP-HSPA2 fusion protein, 100 kDa. Experiments were repeated twice for each HSPA2 model (HSPA2-knockout or GFP-tagged). Representative immunoblots are presented (n = 2). β-actin was used as a protein loading control. Membranes were cut into two (or three) fragments according to the proteins’ molecular weight. For chemiluminescent signal detection X-ray film was used. Original autoradiograms/immunoblots are presented in Fig. S6.
