![Figure 7. PTP1B mRNA and protein levels are increased by IL-4 stimulation. (A) DLBCL cell lines RCK8 or VAL (not shown) were stimulated with IL-4 (100 U/mL) for 3, 6, and 12 hours. RNA was extracted and PTP1B mRNA was measured by real-time RT-PCR in triplicate. In addition, cells lysates were prepared and PTP1B and actin were immunoblotted. (B) To monitor for the effect of IL-4 on protein stability, pulse-chase experiments were performed as described in “Methods” in “Pulse-chase immunoprecipitation assays.” Briefly, RCK8 cells were starved of methionine and then labeled with [;35S]methionine for 2 hours, after which they were either left unstimulated or stimulated with IL-4 (100 U/mL) for up to 24 hours and incorporated [35S]methionine monitored in PTP1B immunoprecipitates by autoradiography. MD indicates mean densitometry of 3 independent experiments. The value at time point 0 was arbitrarily defined as 1. (C) VAL DLBCL cells were transfected with control or STAT6 siRNA. At 48 hours after siRNA transfection, the cells were stimulated with IL-4 for 6 hours. RNA was extracted, and PTP1B and CD23 RNA expression was measured by real-time RT-PCR in triplicate. Cells lysates were immunoblotted for STAT6 and actin. (D) VAL DLBCL cells were grown in complete media supplemented with Ly294002 (10 μM) or PD98059 (30 μM) for 30 minutes. The cells were stimulated with IL-4 for 6 hours, RNA was extracted, and PTP1B RNA expression was measured by real-time RT-PCR in triplicate. (E) RCK8 DLBCL cells were grown in complete media either without or with IL-4 (100 U/mL) for 15 hours. The cells were then stimulated with IL-4 (100 U/mL) for 30 minutes, and cellular lysates were extracted and blotted for pSTAT6, STAT6, PTP1B, and actin. (F) RCK8 DLBCL cells were grown in complete media either without (green) or with IL-4 (100 U/mL; red) for 15 hours. The cells were stained with phycoerythrin-conjugated antihuman IL-4R antibody or appropriate anti isotype control antibody (black) and analyzed by flow cytometry. Representative results from 3 independent experiments are shown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/112/10/10.1182_blood-2008-03-148726/7/zh80210826420007.jpeg?Expires=1735535684&Signature=ulDfVDXZRmhX~koC5sP9ztQucNU3Flo2YX-w6RxaceEwUI-Ym3WLRMNEulAqafwMyEndGr0SvQ7I4ap3~jn75GX1t~1KG7w~b8BWDeIxLgCfYdzsG08FEjCIj~Nduytgzc~We14ifVl2T5QHZZc7K~14iFCzDAAhIIFAcWU6bLM4mnCPZLA7Lc2hVLiuTRpwvRPvVjQlxpo77cCze7HR5TTM-ZqhvdjggM2TFH3gK6PgeVsYYFnK2~CFmR71f6dvyj95NXDTRWZneptCFq-rM31UoS4c4KwbeNaPiW01-eg~MbbP1Nnw8C~7y-WuibCvK7awgx0LHZu06jdY7Vn1AA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
PTP1B mRNA and protein levels are increased by IL-4 stimulation. (A) DLBCL cell lines RCK8 or VAL (not shown) were stimulated with IL-4 (100 U/mL) for 3, 6, and 12 hours. RNA was extracted and PTP1B mRNA was measured by real-time RT-PCR in triplicate. In addition, cells lysates were prepared and PTP1B and actin were immunoblotted. (B) To monitor for the effect of IL-4 on protein stability, pulse-chase experiments were performed as described in “Methods” in “Pulse-chase immunoprecipitation assays.” Briefly, RCK8 cells were starved of methionine and then labeled with [;35S]methionine for 2 hours, after which they were either left unstimulated or stimulated with IL-4 (100 U/mL) for up to 24 hours and incorporated [35S]methionine monitored in PTP1B immunoprecipitates by autoradiography. MD indicates mean densitometry of 3 independent experiments. The value at time point 0 was arbitrarily defined as 1. (C) VAL DLBCL cells were transfected with control or STAT6 siRNA. At 48 hours after siRNA transfection, the cells were stimulated with IL-4 for 6 hours. RNA was extracted, and PTP1B and CD23 RNA expression was measured by real-time RT-PCR in triplicate. Cells lysates were immunoblotted for STAT6 and actin. (D) VAL DLBCL cells were grown in complete media supplemented with Ly294002 (10 μM) or PD98059 (30 μM) for 30 minutes. The cells were stimulated with IL-4 for 6 hours, RNA was extracted, and PTP1B RNA expression was measured by real-time RT-PCR in triplicate. (E) RCK8 DLBCL cells were grown in complete media either without or with IL-4 (100 U/mL) for 15 hours. The cells were then stimulated with IL-4 (100 U/mL) for 30 minutes, and cellular lysates were extracted and blotted for pSTAT6, STAT6, PTP1B, and actin. (F) RCK8 DLBCL cells were grown in complete media either without (green) or with IL-4 (100 U/mL; red) for 15 hours. The cells were stained with phycoerythrin-conjugated antihuman IL-4R antibody or appropriate anti isotype control antibody (black) and analyzed by flow cytometry. Representative results from 3 independent experiments are shown.
