rsk1  (Cell Signaling Technology Inc)

Journal: The Journal of Clinical Investigation

Article Title: RSK1-driven TRIM28/E2F1 feedback loop promotes castration-resistant prostate cancer progression

doi: 10.1172/JCI185119

Figure Lengend Snippet: ( A and B ) RSK1 interacts with TRIM28 in cells. Co-IP was performed in 293T cell lysates expressing MYC-RSK1 along with HA-Flag GFP fusion (as negative control) or HA-Flag TRIM28 ( A ), MYC-TRIM28 along with HA-Flag GFP fusion or HA-Flag RSK1 ( B ) using Flag antibody. The eluted protein was analyzed by immunoblot. ( C – E ) RSK1 phosphorylates TRIM28 at S473 in vitro. Using E.coli -expressed GST-TRIM28 fragments (F1-F2) ( C ), in vitro kinase assay was performed in presence of γ- 32 P–labelled ATP and with the use of an autoradiograph to detect protein phosphorylation ( D ) or in the presence of unlabeled ATP and with the use of immunoblotting to detect pS473-TRIM28 ( E ). ( F and G ) RSK1 phosphorylates TRIM28 at S473 in PCa cells. C4-2B cells transiently expressing MYC-RSK1 along with TRIM28-WT or TRIM28-S473A ( F ); MYC-TRIM28 along with Flag-RSK1-WT, -CA (constitutively active) and -KI (kinase-inactive) were harvested for immunoblot ( G ). ( H and I ) Protein lysates of C42B and DU145 cells with LKO and 2-independent shRSK1 were collected for immunoblot.

Article Snippet: GST-TRIM28 (2 μg fragment 1 and 2) were incubated with 100 ng recombinant active RSK1 (81398, active motif) in 1× kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol [DTT], 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ), plus 5 μCi (γ-32P) ATP (#NEG002A250UC, PerkinElmer) at 30°C for 30 minutes.

Techniques: Co-Immunoprecipitation Assay, Expressing, Negative Control, Western Blot, In Vitro, Kinase Assay, Autoradiography, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: RSK1-driven TRIM28/E2F1 feedback loop promotes castration-resistant prostate cancer progression

doi: 10.1172/JCI185119

Figure Lengend Snippet: ( A – F ) pS473-TRIM28 promotes CRPC growth. LNCaP grown in hormone-depleted medium, C4-2B, and DU145 cells with the indicated treatment were harvested for immunoblot ( A – C ) and analyzed with the colony formation assay for 7–14 days, followed by fixation and crystal violet staining ( D – F ). Quantification was conducted by image J (colony area plugin) and presented as mean ± SEM, n = 3. Statistical analysis was performed using a 2-tailed unpaired Student’s t test, with the Holm-Bonferroni method applied to correct for multiple comparisons. ** P < 0.01, *** P < 0.001. ( G and H ) Xenograft assay was performed by inoculating NSG mice with C4-2B LKO, TRIM28 -KD, and TRIM28 -KD cells rescued by TRIM28-WT and S473A mutant. ( G ) The tumor volume data were presented as mean ± SEM, n = 5. A 1-way ANOVA followed by post hoc multiple comparisons was used for analysis. P value adjustment was performed using Tukey’s Honest Significant Difference (HSD) test. *** P < 0.001. ( H ) Tumor weight data were presented as mean ± SEM, n = 5. Statistical tests performed were 2-tailed unpaired Student’s t-test, with the Holm-Bonferroni method applied to correct for multiple comparisons. * P < 0.05. ( I – K ) Tissue microarray constructed from primary PCa and metastatic CRPC were subjected to IHC staining using anti-pS473–TRIM28 antibody. Representative images and IHC quantification of patient samples at each disease stage were shown. ( L – Q ) RSK1 kinase activity is required for CRPC growth. Hormone-starved LNCaP, C4-2B, and DU145 cells with the indicated treatment were harvested for immunoblot ( L – N ) and subjected to the colony formation assay for 7–14 days ( O – Q ). Quantification was conducted by image J (colony area plugin) and presented as mean ± SEM, n = 3. Statistical analysis was performed using a 2-tailed unpaired Student’s t test, with the Holm-Bonferroni method applied to correct for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: GST-TRIM28 (2 μg fragment 1 and 2) were incubated with 100 ng recombinant active RSK1 (81398, active motif) in 1× kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM β-glycerophosphate, 2 mM dithiothreitol [DTT], 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ), plus 5 μCi (γ-32P) ATP (#NEG002A250UC, PerkinElmer) at 30°C for 30 minutes.

Techniques: Western Blot, Colony Assay, Staining, Xenograft Assay, Mutagenesis, Microarray, Construct, Immunohistochemistry, Activity Assay