HAP1 depletion in leukemic cells inhibits InsP₃-InsP₃R–mediated Ca²⁺ release from the ER. (A) Crude ER fractions in SEM control cells or depleted of HAP1 were isolated,⁴⁴  as described in “Materials and methods.” These were then examined for HAP1, Htt, and InsP₃R levels, and ER enrichment and potential contamination with cytoplasmic proteins by immunoblotting for calnexin, an ER marker, and GAPDH, respectively. S43 denotes the supernatant from the crude ER preparation following centrifugation at 43 000 rpm (Beckman OptimaTM L-90K ultracentrifuge SW-41, as described in “Materials and methods”). Values are means ± standard error of the mean from 3 independent experiments. Calculation was based on the ratios of the levels of HAP1 vs calnexin that were, in turn, based on densitometry of blots with the calnexin value normalized to 1.0. (B) HAP1-depleted cells show reduced formation of the HAP1-Htt-InsP₃R ternary complex. ER fractions from control (left) and HAP1-depleted (right) cells were lysed and subjected to IP using InsP₃R, Htt, or HAP1 antibody. IP samples were resolved by SDS-PAGE and immunoblotted for HAP1, Htt, or InsP₃R. (C) InsP₃-InsP₃R–mediated Ca²⁺ release from the ER is inhibited in HAP1-depleted cells. The chart on the right shows the difference in calcium release from internal stores of control and HAP1-depleted cells upon treatment with 100 nM InsP₃. **P < .05. Measurement of Ca²⁺ release from the ER⁴⁵  upon InsP₃ treatment is described in “Materials and methods.” ER Ca²⁺ release was measured every 2 seconds using a Shimadzu RF 5301PC spectrofluorometer Ca²⁺ imaging at λ_ex = 495 nm and λ_em = 530 nm. Representative data from 1 of 3 independent experiments showing similar result patterns, and indicating reproducibility, are shown in panels A-C.