Figure 3.
FcγRIIB is undetectable by Western blotting in MDM and does not apparently regulate activating FcγR-mediated HIV-1 inhibition. (A) Left: RBL cells transfected with cDNA encoding FcγRIIA or anti-FcγRIIB were western blotted with polyclonal rabbit anti-FcγRIIA or anti-FcγRIIB. Right: B-lymphocytes, monocytes or MDM lysates (10 μg) were western blotted with polyclonal rabbit anti-FcγRIIA or anti-FcγRIIB. (B) RNA from MDMs was analyzed by RT-PCR for expression of FcγRIIA and FcγRIIB transcripts. 0.5 μg of RNA were reverse transcribed. PCR amplification with primers specific to each receptor was performed on sequential ten-fold dilutions of the cDNA mixture. (C) FACS analysis of MDMs stained with F(ab′)2 of mAbs directed against FcγRIIA (IV.3) (solid line) or against the FcγRs IIA, IIB and IIC (AT.10) (dashed line). (D) MDMs were incubated with medium (unstimulated, US), with ICs (IC), with 5μg/106 MDM of irrelevant F(ab′) (C) or with F(ab′)2 derived from IV.3 or AT10 mAbs at decreasing concentrations (5, 1, 0.2, 0.04 μg/106 MDM) and plated on anti-mouse-Fab F(ab′)2 coated wells to cross-link bound F(ab′)2. MDMs were infected with HIV-1 Bal. Results are expressed as percentage of infection (evaluated by p24 levels in supernatants) on day 6 p.i. with respect to unstimulated MDM (p24 = 658 ng/ml). Values are means and SD of three independent wells. Similar results were obtained in experiments performed with MDM from three different donors.