λ phosphatase

ICZ (cis‐4[4‐4‐4[[2‐(2‐4‐dichlorophenyl)‐2‐(1H‐1,2,4,triazol‐1‐methyl)‐1,3‐dioxolan‐4‐yl]‐1‐piperazinyl]phenyl]‐2,4‐dihydro‐2‐(1‐methyl‐propyl)‐3H‐1,2,4‐triazol‐3‐one; MW: 705.64) and ketoconazole were purchased from Sigma‐Aldrich (catalogue numbers #I6657 and #K1003, respectively, St‐Louis, MO, USA), while OSW‐1 ((3β,16β)‐3,17‐dihydroxy‐22‐oxocholest‐5‐en‐16‐yl‐2‐O‐acetyl‐3‐O‐[2‐O‐(4‐methoxybenzoyl)‐β‐D‐xylopyranosyl]‐α‐L‐arabinopyranoside) was from Alfa Chemistry (#ACM145075816, Ronkonkoma, NY, USA). The hydroxy‐ICZ (H‐ICZ), thapsigargin, imipramine and 25‐HC were all purchased from Cayman Chemical (#22576, #10522, #15890 and #11097, respectively, Ann Arbor, MI, USA). Stock solutions were prepared at 10 mM in dimethyl sulfoxide (DMSO) (VWR International, Radnor, PA, USA) for ICZ, H‐ICZ, ketoconazole and thapsigargin; 100 mM for imipramine; 20 mM for 25‐HC; and 1 mM for OSW‐1. Filipin complex from Streptomyces filipinensis was obtained from Sigma‐Aldrich (#F9765), and the stock solution prepared at 25 mg/ml in DMSO. λ‐phosphatase was also purchased from Sigma‐Aldrich (#P9614) at a stock solution of 400,000 units (U) per ml.

ICZ (cis-4[4-4-4[[2-(2-4-dichlorophenyl)-2-(1H-1,2,4,triazol-1-methyl)-1,3-dioxolan-4-yl]-1-piperazinyl]phenyl]-2,4-dihydro-2-(1-methyl-propyl)-3H-1,2,4-triazol-3-one; MW: 705.64) and nocodazole were purchased from Sigma-Aldrich (catalog numbers (#) I6657 and #M1404, respectively). H-ICZ and Sotrastaurin were purchased from Cayman Chemical (#22576 and #16726, respectively) and DNS from Santa Cruz Biotechnology (#sc-202592). The chemical drugs PRR851 and PRR846 were synthetized by one of our labs as described^{38 (link)}. All drugs were diluted in dimethyl sulfoxide (DMSO; VWR International) and used at a final concentration of 10 µM for ICZ, H-ICZ, PRR851, and PRR846, 80 µM for DNS, or 1 µM for nocodazole. λ-phosphatase was purchased from Sigma-Aldrich (#P69614) at a stock solution of 400,000 units (U) per ml.

Fat body from PP2 stages was collected and extracted for total proteins in RIPA lysis buffer, which were further treated with λ-phosphatase (Sigma-Aldrich, Darmstadt, Germany, P9614) according to the manufacturer’s instructions. After treatment, the total proteins were subjected to Western blots analysis detected by BmTip60 antibody.