Figure 4
Figure 4. IFNα and mutant signaling. (A) Macrophages were pretreated or not with anti-IFNAR (CD118) for 20 minutes and treated with parental IFNα or SDM1 for 4 hours. Levels of APOBEC3A for SDM1 compared with parental IFNs *P < .01. APOBEC3A transcription by RT-PCR shows that blockade of IFNAR2 reduces APOBEC3A gene expression, **P < .01. (B) Macrophages were exposed to HIV 2 hours, washed and incubated with CD118 antibody or isotype control for 20 minutes. IFNα21b, IFNα2c SDM1 were then added once at 1 ng/mL and supernatants collected at day 13 after infection and tested for p24 (*P < .01, HIV alone vs IFN-treated). (C) Macrophages were treated with 1 ng/mL of IFNα21b, IFNα2c, or SDM1 in the presence or absence of excess soluble IFNAR2. *P < .05 SDM1 vs 21B and 2C. Inset: Whole cell lysates from macrophages treated with IFNα2c, SDM1 and SDM2 were analyzed for P-JAK1 by Western blot. Representative assay n = 2. (D) Whole cell protein lysates collected after 15-minute exposure to IFNα (0.1 ng/mL) were examined for STAT activation. Levels of STAT1 phosphorylation were analyzed by Western blot using an anti-pSTAT1 antibody. Membranes were then probed with STAT1 antibody to confirm equivalent protein loading. Representative data, n = 4. (E) STAT2 activation was detected 15 minutes after IFN (1 ng/mL) by immunoblotting using pSTAT2 and STAT2 antibodies or β-actin (n = 3). (F) Enhanced phosphorylation of STAT3, STAT5, and STAT6 after treatment with indicated IFNα constructs (1 ng/mL) for 20 minutes (n = 3). (G) Whole cell lysates from macrophages exposed to medium alone, IFNα2c, SDM1, and SDM2 for the indicated time points were analyzed by Western blot for pSTAT1 (Y), pSTAT3 (Y), pSTAT3 (S), pSTAT6 (Y) and tubulin. (H) Macrophages were treated with JAK inhibitor I for 1 hour before addition of IFNα and cell lysates analyzed for pSTAT1 or (I) pSTAT3 by Western blot. Interference with IFNAR-associated JAK suppresses STAT1 and STAT3 phosphorylation when compared with macrophages not treated with inhibitor. Representative data, n = 3. (J) Total RNA analyzed by RT-PCR for APOBEC3A transcription in macrophages pretreated with JAK inhibitor 1 hour prior to 4-hour IFNα. Inhibition of JAK almost completely abrogates APOBEC3A transcription. Representative data, n = 3, *P < .003. Levels of APOBEC3A for SDM1 compared with parental IFNs, **P < .01. Data represent mean + SEM.

IFNα and mutant signaling. (A) Macrophages were pretreated or not with anti-IFNAR (CD118) for 20 minutes and treated with parental IFNα or SDM1 for 4 hours. Levels of APOBEC3A for SDM1 compared with parental IFNs *P < .01. APOBEC3A transcription by RT-PCR shows that blockade of IFNAR2 reduces APOBEC3A gene expression, **P < .01. (B) Macrophages were exposed to HIV 2 hours, washed and incubated with CD118 antibody or isotype control for 20 minutes. IFNα21b, IFNα2c SDM1 were then added once at 1 ng/mL and supernatants collected at day 13 after infection and tested for p24 (*P < .01, HIV alone vs IFN-treated). (C) Macrophages were treated with 1 ng/mL of IFNα21b, IFNα2c, or SDM1 in the presence or absence of excess soluble IFNAR2. *P < .05 SDM1 vs 21B and 2C. Inset: Whole cell lysates from macrophages treated with IFNα2c, SDM1 and SDM2 were analyzed for P-JAK1 by Western blot. Representative assay n = 2. (D) Whole cell protein lysates collected after 15-minute exposure to IFNα (0.1 ng/mL) were examined for STAT activation. Levels of STAT1 phosphorylation were analyzed by Western blot using an anti-pSTAT1 antibody. Membranes were then probed with STAT1 antibody to confirm equivalent protein loading. Representative data, n = 4. (E) STAT2 activation was detected 15 minutes after IFN (1 ng/mL) by immunoblotting using pSTAT2 and STAT2 antibodies or β-actin (n = 3). (F) Enhanced phosphorylation of STAT3, STAT5, and STAT6 after treatment with indicated IFNα constructs (1 ng/mL) for 20 minutes (n = 3). (G) Whole cell lysates from macrophages exposed to medium alone, IFNα2c, SDM1, and SDM2 for the indicated time points were analyzed by Western blot for pSTAT1 (Y), pSTAT3 (Y), pSTAT3 (S), pSTAT6 (Y) and tubulin. (H) Macrophages were treated with JAK inhibitor I for 1 hour before addition of IFNα and cell lysates analyzed for pSTAT1 or (I) pSTAT3 by Western blot. Interference with IFNAR-associated JAK suppresses STAT1 and STAT3 phosphorylation when compared with macrophages not treated with inhibitor. Representative data, n = 3. (J) Total RNA analyzed by RT-PCR for APOBEC3A transcription in macrophages pretreated with JAK inhibitor 1 hour prior to 4-hour IFNα. Inhibition of JAK almost completely abrogates APOBEC3A transcription. Representative data, n = 3, *P < .003. Levels of APOBEC3A for SDM1 compared with parental IFNs, **P < .01. Data represent mean + SEM.

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