American Society of Hematology

Figure 2

$Figure 2. FcγRIIa triggers PLCγ1 activity, PLCγ1 dependent Ca2+ signals, and PKC activity in U937. (A) Immunoblot analysis was used to assay PLCγ1 translocation after FcγRIIa activation by antibody cross-linking in U937. A comparison is shown between a nuclear-free membrane-fraction (top panels) with the cytosolic fraction (bottom panels) probed with an anti-PLCγ1 antibody. TLR-4 and ARF were analyzed as loading controls for the membrane fraction and cytosolic fractions, respectively. (B) U937 stained for PLCγ1 after FcγRIIa cross-linking using antibody clone 3D3 over 5 minutes. Scale bar indicates 20 μm. (C) U937 pretreated with anti-PLCγ1 oligonucleotides down-regulate PLCγ1. Immunoblot analysis of untreated cells, treated cells (a.s.PLCγ1), and scrambled oligo (a.s.scrambled) as a control. α-Tubulin was used as a loading control. (D) FcγRIIa signals through PLCγ1. InsP3 generation was used as a readout for PLCγ1 activation. Basal controls compared with FcγRIIa activation (XL FcγRIIa) and with cells pretreated with antisense oligo (XL FcγRIIa a.s.PLCγ1) or scrambled oligo (XL FcγRIIa a.s.scramb.). (E) PLCγ1 activation by FcγRIIa is linked to cytosolic calcium signaling in U937. Cytosolic calcium signals in U937 were assayed by cuvette fluorimetry after FcR aggregation (untreated XL FcγRI or untreated XL FcγRIIa) compared with cells pretreated with antisense oligo against PLCγ1 (a.s.PLCγ1 XL FcγRI or a.s.PLCγ1 XL FcγRIIa) or a scrambled oligo control (a.s.scramb. XL FcγRIIa). (F) PKC activity in the presence of Ca2+ was measured as the phosphorylation rate (pmol/min) in samples from whole cell lysates after aggregation of FcγRI (labeled XL FcγRI) or FcγRIIa (XL FcγRIIa). (G) PKC activity in the absence of Ca2+ was similarly measured. (H) FcγRI-mediated PKC isoform translocation. Immunoblot analysis of different PKC isoenzymes translocated to the nuclear-free membrane fraction after antibody-mediated FcγRI aggregation over 10 minutes. (I) FcγRIIa-mediated PKC isoform translocation similarly measured in nuclear-free membrane fractions. Immunoblots and calcium traces shown are typical from at least 3 separate experiments. Graphic data represent mean ± SD from 3 experiments.$

FcγRIIa triggers PLCγ1 activity, PLCγ1 dependent Ca²⁺ signals, and PKC activity in U937. (A) Immunoblot analysis was used to assay PLCγ1 translocation after FcγRIIa activation by antibody cross-linking in U937. A comparison is shown between a nuclear-free membrane-fraction (top panels) with the cytosolic fraction (bottom panels) probed with an anti-PLCγ1 antibody. TLR-4 and ARF were analyzed as loading controls for the membrane fraction and cytosolic fractions, respectively. (B) U937 stained for PLCγ1 after FcγRIIa cross-linking using antibody clone 3D3 over 5 minutes. Scale bar indicates 20 μm. (C) U937 pretreated with anti-PLCγ1 oligonucleotides down-regulate PLCγ1. Immunoblot analysis of untreated cells, treated cells (a.s.PLCγ1), and scrambled oligo (a.s.scrambled) as a control. α-Tubulin was used as a loading control. (D) FcγRIIa signals through PLCγ1. InsP₃ generation was used as a readout for PLCγ1 activation. Basal controls compared with FcγRIIa activation (XL FcγRIIa) and with cells pretreated with antisense oligo (XL FcγRIIa a.s.PLCγ1) or scrambled oligo (XL FcγRIIa a.s.scramb.). (E) PLCγ1 activation by FcγRIIa is linked to cytosolic calcium signaling in U937. Cytosolic calcium signals in U937 were assayed by cuvette fluorimetry after FcR aggregation (untreated XL FcγRI or untreated XL FcγRIIa) compared with cells pretreated with antisense oligo against PLCγ1 (a.s.PLCγ1 XL FcγRI or a.s.PLCγ1 XL FcγRIIa) or a scrambled oligo control (a.s.scramb. XL FcγRIIa). (F) PKC activity in the presence of Ca²⁺ was measured as the phosphorylation rate (pmol/min) in samples from whole cell lysates after aggregation of FcγRI (labeled XL FcγRI) or FcγRIIa (XL FcγRIIa). (G) PKC activity in the absence of Ca²⁺ was similarly measured. (H) FcγRI-mediated PKC isoform translocation. Immunoblot analysis of different PKC isoenzymes translocated to the nuclear-free membrane fraction after antibody-mediated FcγRI aggregation over 10 minutes. (I) FcγRIIa-mediated PKC isoform translocation similarly measured in nuclear-free membrane fractions. Immunoblots and calcium traces shown are typical from at least 3 separate experiments. Graphic data represent mean ± SD from 3 experiments.

This Feature Is Available To Subscribers Only