phosphorylated bad (pbad) antibody  (Cell Signaling Technology Inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: PKA inhibition is a central step in D,L-methadone-induced ER Ca 2+ release and subsequent apoptosis in acute lymphoblastic leukemia

doi: 10.3389/fcell.2024.1388745

Figure Lengend Snippet: D,L-methadone-induced ER Ca 2+ release in leukemic cells is associated with downregulation of PLCβ3 Ser1105 inhibitory phosphorylation and BAD phosphorylation at Ser118. (A–C) Lysates of SEM cells pre-treated (or not pre-treated; A) with forskolin (B) or 14–22 amide (myr) (C) then treated with D,L-methadone were subjected to SDS-PAGE and immunoblotting for pSer1105-PLCβ3 and total PLCβ3, and/or pSer118-BAD and total BAD. In C, lysates of cells treated with D,L-methadone for 120 s were used. The numbers under the pSer1105-PLCβ3 and pSer118-BAD bands represent the relative intensity ratios of pSer1105-PLCβ3 or pSer118-BAD vs. total PLCβ3 or BAD bands, respectively, with values at time 0 normalized to 1. Representative blots are from one of three independent experiments (n = 3) showing similar results.

Article Snippet: RPMI 1640 (11875093), Opti-MEM reduced serum (31985062), MEMα (12561056), penicillin-streptomycin (15140122), phosphorylated Ser118 BAD (PA5-12550) and Mag-Fluo-4 a.m. (M14206) were from ThermoFisher Scientific (Burlington, ON, Canada).

Techniques: SDS Page, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: PKA inhibition is a central step in D,L-methadone-induced ER Ca 2+ release and subsequent apoptosis in acute lymphoblastic leukemia

doi: 10.3389/fcell.2024.1388745

Figure Lengend Snippet: Proposed mechanism for D,L-methadone-induced OPRM1-mediated apoptosis in leukemic cells. (A) has shown that D,L-methadone induces leukemic cell apoptosis through the G αi -AC-↓[cAMP] i -caspase pathway (in red). Our previous studies showed that D,L-methadone specifically stimulates the opioid receptor mu1 subtype, OPRM1, in leukemic cells ; D,L-methadone activation of OPRM1 causes increased [Ca 2+ ] i by enhancing IP3R-mediated ER Ca 2+ release and decreasing Ca 2+ efflux, upregulating the Ca 2+ -mediated calpain-1-Bid-cyt C-caspase-3/12 apoptotic pathway (in black) . Together, these findings point to two D,L-methadone-induced OPRM-mediated apoptotic pathways: (i) G αi -AC-↓[cAMP] i -caspase pathway and (ii) ↑[Ca 2+ ] i -calpain-1-Bid-cyt C-caspase-3/12 pathway . Since activation of opioid receptors causes G βγ -mediated ER Ca 2+ release via PLC in neuroblastoma cells and leukocytes ( ; ), in the latter pathway, we proposed the involvement of G βγ (in green) in D,L-methadone-induced ER Ca 2+ release in leukemic cells . (B) In the current study, we demonstrate that D,L-methadone-induced ER Ca 2+ release in ALL cells results from G αi -dependent downregulation of AC and cAMP. PKA inhibition elicits ER Ca 2+ release and overrides D,L-methadone-induced ER Ca 2+ release, indicating that PKA inhibition is a central step in D,L-methadone-induced ER Ca 2+ release. This is consistent with decrease in PKA-dependent (i) inhibitory phosphorylation of PLCβ3 at Ser1105 that leads to PLCβ3 activation and ER Ca 2+ release, and (ii) BAD Ser118 phosphorylation, which ultimately result in caspase activation and apoptosis. Thus, D,L-methadone-induced ER Ca 2+ release and subsequent apoptosis in ALL cells is mediated by G αi -dependent downregulation of the AC-cAMP-PKA-PLCβ3/BAD pathway. Small molecular activators/inhibitors shown in red inhibit ER Ca 2+ release; 14–22 amide (myr) shown in green induces ER Ca 2+ release.

Article Snippet: RPMI 1640 (11875093), Opti-MEM reduced serum (31985062), MEMα (12561056), penicillin-streptomycin (15140122), phosphorylated Ser118 BAD (PA5-12550) and Mag-Fluo-4 a.m. (M14206) were from ThermoFisher Scientific (Burlington, ON, Canada).

Techniques: Activation Assay, Inhibition

Journal: Acta Pharmaceutica Sinica. B

Article Title: Novel dual inhibitor for targeting PIM1 and FGFR1 kinases inhibits colorectal cancer growth in vitro and patient-derived xenografts in vivo

doi: 10.1016/j.apsb.2022.07.005

Figure Lengend Snippet: HCI-48 inhibits the expression level of proteins in the PIM1 and FGFR1 signaling pathways in patient-derived xenograft tumor samples. (A) The expression of Ki-67, PIM1, p-BAD, P27, FGFR1, p-FGFR1, p-STAT3 or p-AKT was examined by IHC analysis in patient-derived xenograft tumors of HJG172 ( n = 10) and HJG194 ( n = 7). (B) The expression of Ki-67, PIM1, p-BAD, P27, FGFR1, p-FGFR1, p-STAT3 or p-AKT was quantified from 4 separate areas on each slide and an average in HJG172 ( n = 5) and HJG194 ( n = 6) in vehicle- and HCI-48-treated groups. (C) The effect of HCI-48 on PIM1 and FGFR1 signaling in patient-derived xenograft tumors of HJG172 and HJG194 assessed by western blot analysis ( n = 4). (D) The effect of HCI-48 on the gene expression of Pim1 and Fgfr1 signaling in patient-derived xenograft tumors of HJG172 and HJG194 was assessed by qRT-PCR analysis ( n = 4). Data are shown as mean ± SD. The asterisks (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001) indicate a significant decrease in treated tissues compared to untreated controls.

Article Snippet: Primary antibodies for the following proteins were purchased from Cell Signaling Technology (Danvers, MA, USA): BAD (1:1000), phosphorylated BAD ( p -BAD, 1:1000), P21 (1:1000), P27 (1:1000), phosphorylated FGFR ( p -FGFR, 1:1000), FGFR1 (1:1000), STAT3 (1:1000), phosphorylated STAT3 ( p -STAT3, 1:1000), AKT (1:1000), phosphorylated AKT ( p -AKT, 1:1000), cyclin A2 (1:1000), PARP (1:1000) and cleaved PARP (1:1000), caspase 3 (1:1000), cleaved caspase 3 (1:1000), caspase 7 (1:1000), cleaved caspase 7 (1:1000), phosphorylated Histone H3 ( p -Histone H3, 1:1000).

Techniques: Expressing, Protein-Protein interactions, Derivative Assay, Western Blot, Gene Expression, Quantitative RT-PCR