human embryonic kidney wt hek 293 cells  (ATCC)

Journal: bioRxiv

Article Title: Rap1b Activates Endosomal AC9 to Drive the Second cAMP Wave

doi: 10.64898/2026.05.06.723328

Figure Lengend Snippet: (A) Schematic representation of the AC9(1037)AC2 chimera, an AC9 backbone carrying the AC2 C2 domain. (B) Representative confocal immunofluorescence micrographs (anti–HA) showing the subcellular localization of the HA–tagged AC9(1037)AC2 chimera at rest and after ISO (30nM) stimulation. Dynasore (dyna; 70μM) was used to block endocytosis–related morphological changes. (C) Real–time cytosolic cAMP measurements in HC–1 cells expressing the AC9(1037)AC2 chimera and the H188 FRET sensor during ISO (30nM) stimulation followed by forskolin and IBMX (F+I; FK 20μM, IBMX 250μM). Traces represent normalized FRET ratios (R/R₀). (D) Dose–response (DR) effects of CAP1 overexpression (CAP1) or knockdown (sh–CAP1) (left) and Rap1b modulation by constitutively active Rap1b G12V or the GTPase–activating protein Rap1 GAP (right) on cAMP production in HC–1 cells expressing the AC9(1037)AC2 chimera stimulated with ISO (30nM). ΔR/R₀ (%) FRET responses were measured with the H188 sensor. (E) Microscale thermophoresis (MST) analysis of AC9–YFP binding to purified His–Gαs (left) or His–Rap1b–GTPγS (right) in vitro . (F) Representative pull–down assay showing interaction between Rap1b and the AC9 C2a domain. HEK293 cells were transfected with HA–Rap1b G12V , and lysates were incubated with Ni–NTA agarose beads loaded with His–tagged AC9 C2a. Complex formation was detected by immunoblotting with an HA–specific antibody. Representative experiment (n = 3). (G) MST analysis of His–GFP–Rap1b G12V binding to purified His–C1a or His–C2a domains in vitro . (H) Schematic representation of CAP1–Rap1b binding to the AC9 C2 domain. Traces (panel C) are shown as mean ± SEM; n ≥ 8 cells from ≥ 3 independent experiments. Significance was assessed at t = 20 min after ISO (vertical line) by one-way ANOVA with Dunnett’s multiple-comparisons test versus vector control. Absence of asterisks denotes non-significance. DR data (panels D, E, G); pooled from n=3 independent experiments) were fit by nonlinear regression to a four–parameter logistic equation to obtain EC₅₀ values with 95% confidence intervals (CI). Differences (vs. vector) were assessed by extra–sum–of–square; p-values reported in Fig. S2E (panel D). EC₅₀ values were 6.0 × 10⁻⁷ M for AC9::Gαs and 1.27 × 10⁻⁶ M for AC9::Rap1b (p < 0.0001) (panel E). EC₅₀ values were 6.56 × 10⁻⁵ M for C1a and 9.66 × 10⁻⁷ M for C2a (p < 0.0001) (panel G).

Article Snippet: The rat hepatoma clonal cell line HC–1 (kindly provided by Dr. Elliot Ross, University of Texas Southwestern Medical Center), human embryonic kidney WT HEK–293 cells (ATCC CRL–1573; Manassas, VA), and HEK–293 ΔGαs (kindly provided by Asaka Inoue, Tohoku University, Japan) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; MT10013CM) supplemented with 10% fetal bovine serum (FBS; FB12999102), penicillin plus streptomycin (100 IU/mL and 100 μg/mL, respectively; 15140122), and L–glutamine (2 mM; 25030164) (All from Thermo Fisher Scientific).

Techniques: Immunofluorescence, Blocking Assay, Expressing, Over Expression, Knockdown, Microscale Thermophoresis, Binding Assay, Purification, In Vitro, Pull Down Assay, Transfection, Incubation, Western Blot, Plasmid Preparation, Control