FIG. 2.

Western blot analysis of anti-SSTR2 monoclonal antibodies against GST-SSTR2 ECD fusion protein and MED cell lines. (A) MAbs A5, C9, C10, and H5 perform nearly identically against GST-SSTR2 ECD protein (e) and D283 MED cell lysate (c), specifically detecting GST-SSTR2 ECD at the projected size of ∼38 kDa (arrow) and the glycosylated SSTR2 in the D283 MED cell lines. GST alone was unreactive (data not shown). Bands detected in lysates represent differentially glycosylated forms, including the nonglycosylated precursor (∼35–40 kDa⁽²⁶⁾). IgG_2a was used as an irrelevant IgG negative primary control, here showing no reactivity. (B) Treatment of the D341 MED cell lysate (20 mg) was carried out to show partial and full deglycosylation of SSTR2 molecules. Lane 1 shows the untreated cell lysate. Lane 2 (25,000 U of PNGase F) and lane 3 (75,000 U of PNGase F) show how varying the amount of PNGase F enzyme during treatment results in varying degrees of deglycosylation. The full-length SSTR2 with the projected mass of ∼83 kDa is visible in the untreated sample, but not visible when treated with the deglycosylation enzyme. The projected size of fully deglycosylated SSTR2 is ∼35–40 kDa, and a band of appropriate size appears, as seen in Lane 3.