![Fig. 6. IL-8 stimulation of bone marrow–derived myeloid precursor cells activates CKLiK. / (A) Ca++ response of 32D cells to hIL-8 (10−7mol/L). Cells were incubated with INDO-am for 45 minutes and washed twice. [Ca++]i concentrations were measured by dual excitation at a wavelength of 340 nm and detected at 390 nm using a Hitachi F4500 fluorescence spectrophotometer. Digitonin was added for 100% [Ca++]i as indicated. (B) IL-8 induces CREB phosphorylation in 32D cells stably expressing wild-type HA_CKLiK. Cells were starved for 4 hours and stimulated with IL-8 (10−7 mol/L) for indicated time periods. HA-tagged CKLiK was immunoprecipitated and kinase assays performed in the absence of Ca++/calmodulin using CREM as a substrate. Ionomycin-treated and untransfected 32D cells were used as positive and negative controls, respectively. Data represent 1 of at least 3 independent experiments. (C) W7 inhibits CKLiK kinase activity. HA_CKLiK stable 32D cells were starved and treated for 15 minutes with or without the calmodulin inhibitor W7 prior to IL-8 (10−mol/L) stimulation. Immunocomplex kinase assays were performed as in (B).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/96/9/10.1182_blood.v96.9.3215/5/h82100292006.jpeg?Expires=1722721377&Signature=t6dlRLuLNNcyVewZ7UfgcbilUX3ow6-4DRLAYqMr44HrTlMZ7MQmysxQFrkO-k0OCfI2VHieLggKhxA5qF-ql29BohxCbvmlmolmsV5WmUBCJJU6Hh2pp-hmg0i~cuhLfIekRPTpnJnoamBu9D4qI40s6ZeG0JbkdOWZ9XiwHdentkOT5F~DSBr0ifTsh3WQm99SZe0SW4qZueG0Kwid0IjjTlGb4P-fhgI1KQs2dhqy8yDtpA7Vpci3jm-Rfu0WxZHjXaS1NKEcBxk8vhANM0zeJAIhKLNYND~bhfok~dgDHN0dHwLBQg~I42wJIjo3Fi7NyeKENHflsMjdruliLw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
IL-8 stimulation of bone marrow–derived myeloid precursor cells activates CKLiK.
(A) Ca++ response of 32D cells to hIL-8 (10−7mol/L). Cells were incubated with INDO-am for 45 minutes and washed twice. [Ca++]i concentrations were measured by dual excitation at a wavelength of 340 nm and detected at 390 nm using a Hitachi F4500 fluorescence spectrophotometer. Digitonin was added for 100% [Ca++]i as indicated. (B) IL-8 induces CREB phosphorylation in 32D cells stably expressing wild-type HA_CKLiK. Cells were starved for 4 hours and stimulated with IL-8 (10−7 mol/L) for indicated time periods. HA-tagged CKLiK was immunoprecipitated and kinase assays performed in the absence of Ca++/calmodulin using CREM as a substrate. Ionomycin-treated and untransfected 32D cells were used as positive and negative controls, respectively. Data represent 1 of at least 3 independent experiments. (C) W7 inhibits CKLiK kinase activity. HA_CKLiK stable 32D cells were starved and treated for 15 minutes with or without the calmodulin inhibitor W7 prior to IL-8 (10−mol/L) stimulation. Immunocomplex kinase assays were performed as in (B).
