polyclonal ip3r1 antibody  (Synaptic Systems)

Journal: Cellular and Molecular Life Sciences

Article Title: Cdk5 regulates IP3R1-mediated Ca 2+ dynamics and Ca 2+ -mediated cell proliferation

doi: 10.1007/s00018-022-04515-8

Figure Lengend Snippet: Cdk5 associates with and phosphorylates IP3R1 at Ser 421 . A Cdk5 associates with IP3R1. Lysates of wt and Cdk5 −/− MEFs were subjected to immunoprecipitation (IP) using IP3R1 antibody. The IPs were resolved by 4–20% gradient SDS-PAGE and then immunoblotted for IP3R1 and Cdk5 (left panel). To assess the specificity of the IP3R1 antibody, lysates of wt MEFs were blotted (right panel) with antibody blocked (lane 2) or not blocked (lane 1) with the peptide antigen that was used to raise the antibody. Lanes 3 and 4 represent IP control using normal IgG. B Cdk5 specifically phosphorylates IP3R1 at Ser 421 . Lysates of wt and Cdk5 −/− MEFs were subjected to SDS-PAGE and then immunoblotted for IP3R1 phosphoSer 421 and phosphoThr 799 , IP3R1, Cdk5 and tubulin. Tubulin blot was used as loading control. Representative blots are from one of four independent experiments ( n = 4) showing similar results. Ratios of levels of IP3R1 phosphoSer 421 (middle panel) and phosphoThr 799 (right panel) vs total IP3R were calculated following densitometric analysis of blots using NIH Image J 1.61. Standard deviations were calculated based on the ratios obtained from the four independent sets of experiments. Values from wt MEFs were normalized to 1.0. * p < 0.05. ns: not significant. C Shows the specificity of the IP3R1 phosphoSer 421 antibody. Lysates of wt MEFs depleted of endogenous IP3R1, but expressing exogenous IP3R1 S 421 A (res) were subjected to immunoblotting for IP3R1 phosphoSer 421 , IP3R1, and Cdk5. GAPDH blot was used as loading control

Article Snippet: The polyclonal antibodies against the two IP3R1 phosphopeptides, MLKIGTpS 421 VKEDKE and HVDRDPQEQVpT 799 PVK, were generated by GL Biochem.

Techniques: Immunoprecipitation, SDS Page, Control, Expressing, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Cdk5 regulates IP3R1-mediated Ca 2+ dynamics and Ca 2+ -mediated cell proliferation

doi: 10.1007/s00018-022-04515-8

Figure Lengend Snippet: IP3R1 loss inhibits the ATP-induced increase in [Ca 2+ ] cyt in Cdk5 −/− MEFs. A Lysates of cells transfected with IP3R1 siRNA #1 or #2 for 48 h were resolved by SDS-PAGE and immunoblotting for IP3R1 and Cdk5. Tubulin blot was used to assess protein loading. Representative blots are from one of three independent experiments showing similar result are shown. B wt and Cdk5 −/− MEFs transfected with IP3R1 siRNA #1 or #2, loaded with Fluo-4 AM, and treated with 1 µM ATP were analyzed for [Ca 2+ ] cyt transients by single-cell Ca 2+ imaging analyses in Ca 2+ -free buffer. Data are means of Ca 2+ signal traces from 20 cells and are from one of three independent experiments showing similar results. [Ca 2+ ] cyt transients were further analyzed by measuring their peak amplitudes ( C ) and calculating the areas under the curve which begin immediately after addition of 1 mM ATP and ends when the Ca 2+ trace goes back to the baseline level. D Values are means ± SEM from three independent experiments ( n = 3). * and **Denote p < 0.05 and p < 0.01, respectively. E Wt and Cdk5 −/− MEFs were co-transfected with the indicated vector and IP3R1 siRNA #2. Cell lysates (40 µg) were resolved by SDS-PAGE and immunoblotted for IP3R1 and Cdk5. GAPDH blot was used as loading control. F Wt and Cdk5 −/− MEFs co-transfected with the indicated vector and siRNA #2 were loaded with Mag-Fluo-4 AM. ER Ca 2+ release following IP3 treatment was measured by spectrofluorometry. Values, which represent the fold change in peak amplitudes, are means ± SEM from three independent experiments ( n = 3). * p < 0.05

Article Snippet: The polyclonal antibodies against the two IP3R1 phosphopeptides, MLKIGTpS 421 VKEDKE and HVDRDPQEQVpT 799 PVK, were generated by GL Biochem.

Techniques: Transfection, SDS Page, Western Blot, Imaging, Plasmid Preparation, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Cdk5 regulates IP3R1-mediated Ca 2+ dynamics and Ca 2+ -mediated cell proliferation

doi: 10.1007/s00018-022-04515-8

Figure Lengend Snippet: Proposed model illustrating how Cdk5 phosphorylation of IP3R1 (Ser 421 ) controls IP3R1-mediated internal Ca 2+ release and [Ca 2+ ] cyt (green text and arrow) and how loss of Cdk5 in Cdk5 −/− MEFs affects [Ca 2+ ] cyt and Ca 2+ -mediated processes (black text and arrows). Loss of Cdk5 reduces the phosphorylation of IP3R1 Ser 421 , causing increased IP3R1-mediated Ca 2+ release. Subsequent rise in [Ca 2+ ] cyt increases ROS production, causing increased Nrf2 expression and activity, and increased expression of the NRF2 antioxidant targets such as Prx1 and Prx2. Adequate [Ca 2+ ] cyt permits progression of Ca 2+ -mediated proliferation, but excess levels cause increased cell proliferation

Article Snippet: The polyclonal antibodies against the two IP3R1 phosphopeptides, MLKIGTpS 421 VKEDKE and HVDRDPQEQVpT 799 PVK, were generated by GL Biochem.

Techniques: Phospho-proteomics, Expressing, Activity Assay