Figure 2: mAb SKM9-2 recognizes a sialylated HEG1 on the cell surfaces of mesothelioma cells. | Scientific Reports

Figure 2: mAb SKM9-2 recognizes a sialylated HEG1 on the cell surfaces of mesothelioma cells.

From: HEG1 is a novel mucin-like membrane protein that serves as a diagnostic and therapeutic target for malignant mesothelioma

Figure 2

(a) Representative immunoblotting (n = 2) of SKM9-2 antigen in MPM cell lines. Cell lysates (10 μL per lane) were resolved by SDS-PAGE under non-reducing conditions. (b) Flow cytometric analysis of SKM9-2 antigen on ACC-MESO-4. An irrelevant mAb (2D2, mouse IgG1) was used as a negative control mAb. (c) Elimination of recognition with mAb SKM9-2 by glycosidase treatment. A partially purified sample was treated with glycosidase or proteinase K and resolved by SDS-PAGE under reducing conditions. Similar results were obtained by 3 independent analyses. PNGase F, peptide N-glycosidase F. (d,e,f,g) Purification of SKM9-2 antigen using a Superose 6 Increase 10/300GL, Mono Q 5/50GL, WGA-agarose, or Superose 6 Increase 10/300GL column, respectively. Dot blot results are also shown. Pooled fractions are indicated as a black bar. (h) Western blotting and CBB staining of purified SKM9-2 antigen. (i) Results of Mascot search for purified SKM9-2 antigen. The purified sample was resolved by SDS-PAGE. The CBB-stained band was then cut from the gel and analyzed by mass spectrometry and Mascot search. The highest-scoring hit is shown. (j) Decrease of SKM9-2 antigen by gene silencing of HEG1. Cell lysates (7.5 μL per lane) were resolved by SDS-PAGE under reducing conditions and detected by western blotting using mAb SKM9-2. Similar results were obtained by 3 independent analyses. After detection, the membrane was reprobed and treated with anti-β-actin mAb (AC-15). Control siRNA, MISSION siRNA Universal Negative Control (SIC-001); H1097, H2674, and H3671, HEG1 siRNAs; copGFP, a control lentiviral particles. (k) Representative western blotting (n = 2) of HEG1-transfected HEK293T (HEK293T + HEG1) using mAb SKM9-2. Cell lysates (5 μL per lane) were resolved by SDS-PAGE under reducing conditions. EGFP-transfected HEK293T (HEK293T + EGFP) was used as a negative control. After detection, the membrane was reprobed and treated with anti-β-actin mAb (AC-15).

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