Figure 2

Imaging, immunoblot and RT-PCR analysis of MR protein expression from macrophage cells. In panel A, 43^MR cells were processed for confocal imaging as described in Methods. Cells were fixed in 2% buffered paraformalin for 15 minutes and permeabilized in ice cold methanol for 10 minutes. Cells were then washed and stained with mouse anti-MR directly conjugated to Alexa 488 and rabbit anti-EEA1 antibody followed by goat anti-rabbit Alexa 568 conjugated secondary. Nucleus was stained with TO-PRO 3 for visual reference. (B) RBMM and 43^MR cells (1 x 10⁶) were harvested and lysed in NP-40 lysis buffer for immunoblot analysis. Proteins (10 μg) were resolved on a 7.5% SDS-PAGE gel. Immunoblot analysis was performed with a polyclonal antibody against the MR and HRP-conjugated secondary antibody. Detection of protein was performed by enhanced chemiluminescent assay and exposure of Kodak Bio-Max scientific film. (C) Total mRNA was prepared from 43^MR cells (1 x 10⁶) using Trizol reagent, and reversed transcribed using the First-Strand cDNA kit. The cDNA preparations were then subjected to PCR using the 5’ and 3’ primers described for the 650 bp and 300 bp fragments. The data are representative of three independent experiments.