Expression of PMR1 in primary rat islets and clonal β-cell lines. A: Total RNA was extracted from INS1 and MIN6 cells, rat islets, and rat brain; reverse-transcribed; and amplified using PCR
with primers designed to amplify a 640-bp internal region of PMR1. A negative control (No RT) was performed using non-reverse-transcribed
RNA as a template for PCR. The sequence of the mouse clone is given in the online appendix (Gene Bank accession no. AJ551270).
B: Rat islet protein (70 μg per lane) or whole-cell lysate from INS1 or MIN6 cells (20 μg per lane) was separated on a 7.5%
(wt/vol) polyacrylamide gel, blotted onto Immobilon-P transfer membrane, and probed with anti-PMR1 antibody (1:1,000) in the
absence or presence (as indicated, at 40 μg/ml) of the 17–amino acid peptide used for antibody generation. HRP-conjugated
anti-rabbit IgG secondary antibody (1:80,000; Sigma) was used for visualization.