Journal: Cancer Cell International
Article Title: Regulation of microtubule nucleation in glioblastoma cells by ARF GTPase-activating proteins GIT1 and GIT2 and protein kinase C
doi: 10.1186/s12935-025-03740-y
Figure Lengend Snippet: PKC modulates microtubule nucleation and phosphorylates GIT2. ( A ) Immunoprecipitation experiments using whole-cell extracts from U-251 MG cells and immobilized Ab to PKCα. The blots were probed with Abs to GIT2, PKCα, γ-tubulin (γ-Tb), and calcineurin (Calcin., negative control). Load ( lane 1 ), immobilized Abs without cell extracts ( lane 2 ), precipitated proteins ( lane 3 ), and Ab-free carrier incubated with cell extract ( lane 4 ). ( B ) GST-fusion protein GIT2 (1–124) or GST alone were immobilized on Glutathione-Sepharose and incubated with a whole-cell extract from U-251 MG cells. The blots were probed with Abs to PKCα, γ-tubulin (γ-Tb), calcineurin (Calcin., negative control), and GST. Load ( lane 1 ), GST-fusion protein without cell extract ( lane 2 ), proteins bound to GST-fusion protein ( lane 3 ), and proteins bound to GST alone ( lane 4 ). ( C ) Comparison of the microtubule nucleation rate (EB3 comets/min) in cells with activated PKCs, relative to control cells. Cells were pretreated with DMSO alone (Control), PKC activator PMA (1 μM for 30 min), or the PKC inhibitor Gö6976 (10 μM for 30 min) before adding PMA. Three independent experiments were conducted (at least 17 cells counted in each experiment): Control (n = 75), + PMA (n = 75), + PMA + Gö6976 (n = 77). The bold and thin lines within the dot plots represent mean ± SD. A one-way ANOVA with Sidak’s multiple comparison test was performed to determine statistical significance. ****, p < 0.0001. ( D – E ) Immunoprecipitation experiments with Abs to NG and whole-cell extracts from cells expressing GIT2-NG. ( D ) Kinase assay following immunoprecipitation ( 32 P). Load ( lane 1 ), immobilized Ab without cell extract ( lane 2 ), precipitated proteins ( lanes 3–4 ), and Ab-free carrier incubated with cell extract ( lane 5 ). Cells were pretreated with DMSO alone ( lane 3 ) or with the PKC inhibitor Gö6976 (10 μM for 30 min; lane 4 ). The blot was probed with Ab to NG. The numbers under the blot indicate the relative amounts of phosphorylated GIT2-NG ( 32 P) normalized to Gö6976 untreated cells and the amount of precipitated GIT2-NG in individual samples (fold). Mean ± SD (n = 7). ( E ) Phosphorylation of GIT2-NG by PKC. Immobilized Ab without cell extract ( lane1 ), precipitated proteins ( lanes 2–4 ), and Ab-free carrier incubated with cell extract ( lane5 ). Cells were pretreated with DMSO alone ( lane 3 ) or with the PKC selective inhibitor Gö6976 (10 μM for 30 min; lane 4 ) before incubation with calyculin A (80 nM for 30 min). The blots were probed with Abs to P-Ser in the PKC motif and NG. The numbers under the blot indicate the relative amounts of GIT2-NG phosphorylated on serine (P-Ser in the PKC motif) normalized to Gö6976-untreated cells and the amount of precipitated GIT2-NG in individual samples (fold). Mean ± SD (n = 5). ( F ) Kinase assay after immunoprecipitation experiment with Ab to GIT2 and whole-cell extract ( 32 P). Load ( lane 1 ), immobilized Ab without cell extract ( lane 2 ), precipitated proteins ( lanes 3–4 ), and Ab-free carrier incubated with cell extract ( lane 5 ). Cells were pretreated with DMSO alone ( lane 3 ) or with the PKC inhibitor Gö6976 (10 μM for 30 min; lane 4 ). The blot was probed with Ab to GIT2. The numbers under the blot indicate the relative amounts of phosphorylated GIT2 ( 32 P) normalized to Gö6976-untreated cells and the amount of precipitated GIT2 in individual samples (fold). Mean ± SD (n = 6). ( G ) Relative distribution of proteins in fractions after differential centrifugation of homogenates from GIT2_KD + GIT2-NG cells. Cell fractions were prepared as described in the Materials and Methods section. Cell homogenate ( lane 1 ), pellet P1 ( lane 2 ). Immunoblot analysis was performed with Abs to NG, pericentrin, and actin (loading control). Densitometric quantification of immunoblots is shown on the right. Intensities of corresponding proteins in P1 normalized to loads (relative intensity 1.0). Mean ± SD (n = 4). ( D-G ) A two-tailed, unpaired Student’s t -test was performed to determine statistical significance. ***, p < 0.001; ****, p < 0.0001
Article Snippet: Rabbit Abs directed against calcineurin (2614) and P-Ser in PKC substrate motif (6967) were acquired from Cell Signaling Technology (Danvers, MA, USA).
Techniques: Immunoprecipitation, Negative Control, Incubation, Comparison, Control, Expressing, Kinase Assay, Phospho-proteomics, Centrifugation, Western Blot, Two Tailed Test
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