rab8a  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: Loss of Ric-8A leads to YM-sensitivity of αqAG-QL and increases sensitivity of αqQL to YM. A , TEAD luciferase assay in RIC-8A KO cells transfected with either pcDNA3 (negative control), αqQL, αqAG-QL, and increasing amounts of RIC-8A as indicated. TEAD luciferase 8X-GTIIC reporter and renilla luciferase were cotransfected. Two hours after transfection, media were changed to serum-free and 1 μM YM was added overnight where indicated. Cells were lysed the following day, and luciferase experiments were performed. TEAD luciferase values were normalized to renilla luciferase values for the same well and averaged for each condition. Results are shown as mean ± SD and statistical significance is indicated. (n = 3, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001, two-way ANOVA, Šidák's multiple comparison’s test). αq protein expression was assessed by immunoblotting. B , immunoblot for Ric-8A demonstrating endogenous levels in q/11 KO cells in comparison to exogenously expressed levels in RIC-8A KO cells. Endogenous Ric-8A migrates slightly slower than the plasmid-expressed Ric-8A, and this is indicated by the two lines at the Ric-8A immunoblot. This difference may be due to alternative methionine initiation or slight proteolysis of the exogenously expressed Ric-8A. C , YM IC50 analysis readout by the TEAD luciferase reporter comparing RIC-8A KO and q/11 KO cells transfected with αqQL and treated with YM concentrations ranging from 0.0001 to 1 μM. YM treatments were overnight in serum-free media (n = 3).

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Luciferase, Transfection, Negative Control, Expressing, Western Blot, Comparison, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: Ric-8A only exerts its effects on YM-sensitivity in the context of CA αq, not αq WT. A and B , SRE luciferase reporter assays in RIC-8A KO cells transfected with pcDNA3 (negative control), αqQL, or αq WT, and increasing amounts of RIC-8A where indicated, along with SRE and renilla luciferase plasmids. For αq WT experiments ( B ), m3AChR was coexpressed. Two hours after transfection, media were changed to serum-free and 1 μM YM was added overnight where indicated. Cells were lysed the following day, and luciferase experiments were run. B , to stimulate αq WT, 100 μM carbachol was added 1 h after YM-treatment. SRE values are normalized to respective renilla values for the same well and averaged for each condition. Results are shown as mean ± SD and statistical significance is indicated. (n = 3, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001, two-way ANOVA, Šidák's multiple comparison’s test). C , lysates were immunoblotted to assess αq protein levels. m3AChR, muscarinic acetylcholine m3 receptor; SRE, serum response element.

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Luciferase, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: αqAG-QP YM sensitivity is directly altered by Ric-8A. A , TEAD luciferase assay in RIC-8A KO cells transfected with either pcDNA3 (negative control), αqQP, αqAG-QP, and increasing amounts of RIC-8A as indicated. TEAD luciferase 8X-GTIIC reporter and renilla luciferase were cotransfected. Two hours after transfection, media were changed to serum-free and 1 μM YM was added overnight where indicated. Cells were lysed the following day and luciferase experiments were performed. TEAD luciferase values were normalized to renilla luciferase values for the same well and averaged for each condition. B and C , SRE-luciferase reporter assays in q/11 KO cells transfected with pcDNA3 (negative control), αqQL/P, or αqAG-QL/P, and SRE and renilla luciferase plasmids. Where indicated, increasing amounts of RIC-8A were added . The same protocol was followed as in A. Lysates were immunoblotted to assess αq protein levels ( bottom , B ). SRE values are normalized to their respective renilla value and averaged for each condition. Results are shown as mean ± SD and statistical significance is indicated. (n = 3, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.005, two-way ANOVA, Šidák's multiple comparison’s test). SRE, serum response element.

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Luciferase, Transfection, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: BRET studies reveal that αqAG-QL remains GTP-bound and active in the presence of YM, and Ric-8A can increase the amount of activated αqAG-QL and αqQL. A , schematic depicting bioluminescence resonance energy transfer (BRET) sensors for activated GTP-bound venus-αq interaction with NLuc-GRK2-RH, which would lead to high BRET signal upon addition of luciferase substrate. B and C , HEK293 ( B ) or HEK293 RIC-8A KO cells ( C ) were transfected with 25 ng of Nano-luciferase fused GRK2-RH (donor) and 500 ng of Venus-fused αq (acceptor). 300 ng of Gβ and 100 ng of Gγ were coexpressed. Additionally, RIC-8A KO cells were transfected with 10 ng of RIC-8A where indicated ( C ). The following day, respective samples were treated with 1 μM YM for 2 h and BRET measurements were read 2 min after addition of Nano-glo (Promega). Venus-αq WT values were subtracted as background from all emission ratios (acceptor/donor). Results are shown as mean ± SD and statistical significance indicated (n = 4 in HEK293 and n = 3 in HEK293 RIC-8A KO cells, ∗∗ p < 0.01; ∗∗∗ p < 0.005; ∗∗∗∗ p < 0.0001, two-way ANOVA, Šidák's multiple comparison’s test). D , validation of expression of venus-αq constructs in RIC-8A KO cells with and without Ric-8A and YM. RH, RGS-homology.

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Bioluminescence Resonance Energy Transfer, Luciferase, Transfection, Biomarker Discovery, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: The guanine nucleotide exchange factor Ric-8A regulates the sensitivity of constitutively active Gαq to the inhibitor YM-254890

doi: 10.1016/j.jbc.2025.108426

Figure Lengend Snippet: αqQL and αqAG-QL bind GRK2-RH more strongly in the presence of Ric-8A. A and B , cells were transfected with FLAG-GRK2-RH and either αqQL or αqAG-QL with and without 10 ng of RIC-8A . As negative controls, RIC-8A KO cells were transfected with pcDNA3, αq WT, FLAG-GRK2-RH, αq WT + FLAG-GRK2-RH, or αq WT + FLAG-GRK2-RH + 10 ng of RIC-8A. Three hours after transfection, 1 μM YM was added where indicated overnight. A , cells were lysed the day after transfection, and FLAG beads were used to immunoprecipitate FLAG-GRK2-RH, and the pulldowns and inputs subsequently immunoblotted for the proteins indicated. B , the αq pull-down signal intensities were quantified and normalized to their respective input signal intensity. Results are shown as mean ± SD. Statistical significance is indicated (n = 4, ∗ p < 0.05; ∗∗ p < 0.01, two-way ANOVA, Šidák's multiple comparison’s test). RH, RGS-homology.

Article Snippet: The pERK1/2 (Cat. #9101S) and ERK1/2 (Cat. #4696S) antibodies were from Cell Signaling Technologies. αq (Cat. #13927-1-AP), GAPDH (Cat. #60004-1-Ig), and Ric-8A (Cat. #66625-1-Ig) were from Proteintech.

Techniques: Transfection

Journal: Stem cell research & therapy

Article Title: Autophagy modulation effect on homotypic transfer of intracellular components via tunneling nanotubes in mesenchymal stem cells.

doi: 10.1186/s13287-024-03813-1

Figure Lengend Snippet: Fig. 4 Western blotting (A and B). Data revealed the lack of significant differences in protein levels of autophagy machinery (BCLN1, LC3-II/LC3-I ratio, and p62) in the presence of 15 µM Met, and 3 µM 3-MA after 48 h. Protein levels of Miro1 and 2 remained unchanged in the presence of Met, and 3-MA. The levels of Rab8 and p-FAK related to TNT assembly and vesicle transport were reduced significantly in 3-MA-treated MSCs. Data are expressed as mean ± SD. One-way ANOVA and Tukey post hoc test (n = 3). *p < 0.05; **p < 0.01

Article Snippet: Membranes were blocked using 2% skim milk solution at RT for 1 h, and incubated with primary antibodies overnight at 4 °C as follows; BECLN-1 (Cat no: sc-48,341; Santa Cruz Biotechnology, Inc), LC3 (Cat no: 2775; Cell Signaling Technology, Inc), and P62 (Cat no: sc-10,117; Santa Cruz Biotechnology, Inc), Miro1 (Cat no: sc-398,520; Santa Cruz Biotechnology, Inc), Miro 2 (Cat no: sc-135,387; Santa Cruz Biotechnology, Inc), Rab8 (Cat no: sc-81,909; Santa Cruz Biotechnology, Inc), (Cat no: sc-81,493; Santa Cruz Biotechnology, Inc).

Techniques: Western Blot