Journal: The Journal of Biological Chemistry
Article Title: The store-operated Ca 2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCβ2
doi: 10.1016/j.jbc.2023.102882
Figure Lengend Snippet: TRPC1 and Orai3 are involved in carbachol (CCh)-evoked NF-κB activation in Orai1-KO cells. A, Orai1-KO HEK-293 cells (O1KO) transfected with either shTRPC1 (O1KO (shT1)), shTRPC6 (O1KO (shT6)), shOrai3 (O1KO (shO3)), or scramble plasmid (O1KO) were transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and then stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity of the lysates was measured as described in the section. The luminescence in relative light units (RLUs) of unstimulated O1KO, O1KO(shT1), O1KO(shT6), and O1KO(shO3) HEK-293 cells were 598,259 ± 47,521, 601,025 ± 51,248, 599,987 ± 32,581, and 601,277 ± 39,985, respectively). B, O1KO, O1KO (shT1), and O1KO (shT6) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-TRPC1 ( left panels ) or anti-TRPC6 ( right panels ) antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of TRPC1 and TRPC6 bands was normalized to β-actin and quantified ( bar graph ). C, WT HEK-293 cells (WT), WT cells transfected with shOrai3 (shO3), O1KO cells, O1KO cells transfected with shOrai3 (O1KO (shO3)), and Orai1/Orai2/Orai3 triple-KO cells (TKO) transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity was measured as described in the section. The luminescence in RLUs of unstimulated WT, O1KO, O1KO(shO3), and TKO HEK-293 cells were 610,982 ± 48,875, 607,588 ± 47,510, 592,115 ± 37,514, and 597,323 ± 37,899, respectively. D, WT HEK-293 cells (WT) and WT cells transfected with shOrai3 (shO3) were lysed and subjected to 10% SDS-PAGE and Western blotting with anti-Orai3 antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). E, WT, O1KO, TKO, and O1KO (shO3) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-Orai3. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). For A, from left to right , n = 18, 14, 18, 13, 18, 12, 18, and 13; for C, from left to right , n = 30, 24, 12, 12, 18, 13, 18, 14, 18, and 12; n values correspond to separate experiments. Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). ∗ p < 0.05 and ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as compared with CCh-treated O1KO HEK-293 cells ( A ) or CCh-treated WT HEK-293 cells ( C ). $ p < 0.05, $$ p < 0.01, and $$$$ p < 0 .0001 as compared with their respective control (untreated) cells. HBS, Hepes-buffered saline; HEK-293, human embryonic kidney 293 cell line.
Article Snippet: High-glucose Dulbecco’s modified Eagle’s medium, fetal bovine serum, trypsin, penicillin/streptomycin, TRIzol reagent, qRT–PCR primers, high-capacity complementary DNA reverse transcription kit, SYBR Green PowerUp, rabbit polyclonal anti-TRPC1 antibody (catalog number: PA577303, epitope: amino acids 557–571 of human TRPC1), Clean-Blot IP detection reagent, and SuperSignal West Dura extended duration substrate reagent, and Pierce BCA protein assay kit were purchased from Thermo Fisher Scientific.
Techniques: Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, SDS Page, Western Blot
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![<t>TRPC1</t> and Orai3 are involved in carbachol (CCh)-evoked NF-κB activation in Orai1-KO cells. A, Orai1-KO HEK-293 cells (O1KO) transfected with either shTRPC1 (O1KO (shT1)), shTRPC6 (O1KO (shT6)), shOrai3 (O1KO (shO3)), or scramble plasmid (O1KO) were transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and then stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity of the lysates was measured as described in the section. The luminescence in relative light units (RLUs) of unstimulated O1KO, O1KO(shT1), O1KO(shT6), and O1KO(shO3) HEK-293 cells were 598,259 ± 47,521, 601,025 ± 51,248, 599,987 ± 32,581, and 601,277 ± 39,985, respectively). B, O1KO, O1KO (shT1), and O1KO (shT6) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-TRPC1 ( left panels ) or anti-TRPC6 ( right panels ) antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of TRPC1 and TRPC6 bands was normalized to β-actin and quantified ( bar graph ). C, WT HEK-293 cells (WT), WT cells transfected with shOrai3 (shO3), O1KO cells, O1KO cells transfected with shOrai3 (O1KO (shO3)), and Orai1/Orai2/Orai3 triple-KO cells (TKO) transfected with pNL3.2.NFkB-RE[NlucP/NF-kB-RE/Hygro]. Forty-eight hours later, cells were suspended in HBS containing 1 mM Ca 2+ and stimulated for 5 h with 100 μM CCh or the vehicle (control) and lysed. Luciferase activity was measured as described in the section. The luminescence in RLUs of unstimulated WT, O1KO, O1KO(shO3), and TKO HEK-293 cells were 610,982 ± 48,875, 607,588 ± 47,510, 592,115 ± 37,514, and 597,323 ± 37,899, respectively. D, WT HEK-293 cells (WT) and WT cells transfected with shOrai3 (shO3) were lysed and subjected to 10% SDS-PAGE and Western blotting with anti-Orai3 antibody. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). E, WT, O1KO, TKO, and O1KO (shO3) HEK-293 cells were lysed and then subjected to 10% SDS-PAGE and Western blotting with anti-Orai3. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four separate experiments, and intensity of Orai3 bands was normalized to β-actin and quantified ( bar graph ). For A, from left to right , n = 18, 14, 18, 13, 18, 12, 18, and 13; for C, from left to right , n = 30, 24, 12, 12, 18, 13, 18, 14, 18, and 12; n values correspond to separate experiments. Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn's test). ∗ p < 0.05 and ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as compared with CCh-treated O1KO HEK-293 cells ( A ) or CCh-treated WT HEK-293 cells ( C ). $ p < 0.05, $$ p < 0.01, and $$$$ p < 0 .0001 as compared with their respective control (untreated) cells. HBS, Hepes-buffered saline; HEK-293, human embryonic kidney 293 cell line.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2819/pmc09922819/pmc09922819__gr5.jpg)
