Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47 phox

doi: 10.1073/pnas.2017830117

Figure Lengend Snippet: The slow proliferation of Akt3-expressing cells is caused by ROS, induced by Akt3, via p47phox phosphorylation, and activation of the NADPH oxidase. (A) Phosphorylation of p47phox in Akt1-, Akt2-, and Akt3-expressing cells growing in serum-supplemented media. Akt1-, Akt2-, and Akt3-expressing lung fibroblasts were transduced with wild-type Flag-p47phox. p47phox was immunoprecipitated from cell lysates with an anti-Flag antibody. A Western blot of the immunoprecipitates was probed with an Akt phosphosubstrate antibody (RXXS*/T*). The same immunoprecipitates were probed with the anti-Flag antibody (loading control). (B) Growth curves of p47phox−/− MEFs expressing Akt1 or Akt3 and growing in complete serum-supplemented media. (C) Western blot showing the expression of p53 in cell lysates of Akt1- and Akt3-expressing p47phox−/− MEFs growing in serum-supplemented media. Tubulin was used as the loading control. (D and E) DCFDA- and DHE-detectable ROS in the Akt1- and Akt3-expressing p47phox−/− MEFs were measured by flow cytometry. (F) DHE-detectable ROS levels were measured by flow cytometry, in p47phox−/− MEFs and their derivatives, transduced with the empty vector (pBabe-neo) or with wild-type p47phox (p47phoxR). The wild-type p47phox-rescued cells were also transduced with Akt1 or Akt3 retroviral constructs or with the empty vector (EV). (G) Conservation of the Akt phosphorylation motifs, RXXS/T, on p47phox (Ser304 and Ser328). (H) Phosphorylation of wild type and p47phox mutants in Akt1- and Akt3-expressing cells growing in serum-supplemented media. Akt1- and Akt3-expressing lung fibroblasts were transduced with wild-type Flag-p47phox (WT) or its mutants Flag-p47phoxS304A, Flag-p47phoxS328A, or Flag-p47phoxS304A/S328A double mutant (DM). Cell lysates were immunoprecipitated with the anti-Flag antibody (p47phox), and Western blots of the immunoprecipitates were probed with the Akt phosphosubstrate antibody (RXXS*/T*). Probing immunoprecipitates with anti-Flag antibody were used as the loading control. The same immunoprecipitates probed with anti-Flag were used as the loading control. (I) DHE-detectable ROS, measured by flow cytometry, in p47phox−/− MEFs and their derivatives, transduced with the wild-type p47phox (p47phoxR) or the double phosphorylation site mutant of p47phox (DMp47phoxR). Both the wild type and the mutant p47phox-rescued cells were also transduced with Akt1 or Akt3 retroviral constructs. EV/EV, cells transduced with both empty vectors; R, rescued.

Article Snippet: Akt1-, Akt2-, and Akt3-specific antibodies, as well as antibodies against p53, flag-tag, phosphorylated Akt substrate (RXXS/T), and phosphorylated histone H2AX on Ser139, were purchased from Cell Signaling Technology.

Techniques: Expressing, Activation Assay, Transduction, Immunoprecipitation, Western Blot, Flow Cytometry, Plasmid Preparation, Construct, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

doi: 10.1186/s13046-019-1385-7

Figure Lengend Snippet: Sequences of primers used

Article Snippet: The primary antibodies used were ROCK1(sc-5560), ROCK2(sc-5561), ROCK2(CST-9029), ROCK2(sc-398519), MRE11(CST-4847), NBS1(CST-3001), RAD50(CST-3427), C-PARP (CST-9541), pH2Ax (Calbiochem DR1017), DNA-PK (CST-4602), β-ACTIN (Sigma, clone AC-74), Tubulin (Sigma N6786), RhoC (CST-D40E4), Histone 3 (CST-D1H2), CDK1 (sc-54), pCDK1(CST-9114), pP53-Ser15 (CST-9284), pAKT-Ser473 (CST-9271) and GAPDH (sc-47724).

Techniques: Sequencing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

doi: 10.1186/s13046-019-1385-7

Figure Lengend Snippet: Evaluation of the role of ROCK2 in radiation response in SiHa cells. a Immunoblot analysis of whole cell extracts of SiHa cells showed an upregulation of both ROCK1 and ROCK2 proteins upon irradiation at the indicated time points. b Immunofluorescence analysis also showed that the ROCK2 expression levels were higher in the nuclear compartment as compared to the ROCK1 levels. Scale bar = 10 μm.( n = 3). c Immunoblot analysis of ROCK1 and ROCK2 at different times points in the nuclear extracts of IR SiHa cells ( n = 3). d Graphical representation of flow cytometry analysis showing an increased percentage of cells with ROCK2 expression in the irradiated (IR) SiHa cells as compared to the non-irradiated (NR) control cells. A fold increase of 1.4 is depicted ( p < 0.03; n = 3). e Inhibition of ROCK signaling using Y27632 (10 μM) to determine its effect on cell survival. There was a 30% increase in cell death of the treated cells due to irradiation ( n = 3, * p < 0.037; # n.s). f SiHa cells transfected with 1 μg of pCAG-ROCK2 expression vector and the corresponding empty vector followed by irradiation displayed increased survival. ( p < 0.01, n = 4). g-i SiHa cells treated with saponin (Sap+ve) had comparable cell viability as compared to the untreated control (Sap -ve), #- n.s ( n = 3). g-ii Immunoblot analysis showed that there is reduction in ROCK2 levels but not ROCK1 upon inhibition with the ROCK2 antibody (ROCK2 In ). IgG isotype has been used as control ( n = 3). g-iii Representative histograms depict the increased PI uptake in cells with ROCK2 In as compared to the corresponding isotype control. g-iv Graphical representation of flow cytometry-based analysis of cell survival upon ROCK2 In followed by irradiation shows increased sensitization of SiHa cells to irradiation ( n = 3, p < 0.001). h-i Graphical representation of weights of tumors formed by NR and IR cells (NR tumor = 0.53 g ±0.28; IR tumor = 0.76 g ±0.32, n = 3, p < 0.02). h-ii Representative images of immunofluorescence-based analysis of xenografts derived from NR and IR tumors showed increased expression of ROCK2 in IR tumor derived sections (scale bar = 50 μm)

Article Snippet: The primary antibodies used were ROCK1(sc-5560), ROCK2(sc-5561), ROCK2(CST-9029), ROCK2(sc-398519), MRE11(CST-4847), NBS1(CST-3001), RAD50(CST-3427), C-PARP (CST-9541), pH2Ax (Calbiochem DR1017), DNA-PK (CST-4602), β-ACTIN (Sigma, clone AC-74), Tubulin (Sigma N6786), RhoC (CST-D40E4), Histone 3 (CST-D1H2), CDK1 (sc-54), pCDK1(CST-9114), pP53-Ser15 (CST-9284), pAKT-Ser473 (CST-9271) and GAPDH (sc-47724).

Techniques: Western Blot, Irradiation, Immunofluorescence, Expressing, Flow Cytometry, Inhibition, Transfection, Plasmid Preparation, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

doi: 10.1186/s13046-019-1385-7

Figure Lengend Snippet: Transcriptional analysis highlights enrichment of DNA repair and survival pathway genes in ROCK2 hi cells. a A graphical representation of survival following irradiation in ROCK2 hi cells and ROCK2 lo cells. The viability was determined using the WST1 assay reagent. ROCK2 hi cells showed better survival. Fold ratio survival represented is normalized to that of the ROCK2 lo cells ( n = 4, 1.4-fold, p < 0.03). b A graphical representation of increased migration of ROCK2 hi cells as compared to ROCK2 lo cells sorted based on ROCK2. * p < 0.03 ( n = 3). c GO enrichment analysis of the selected biological processes in the ROCK2 hi cells with a p < 0.05 represented as a graph with -Log 10 P -values plotted on x-axis. d-i String interactome analysis was performed on the set of 51 genes that broadly represented DNA repair, cell cycle apoptosis and cell division. The network of DNA repair proteins formed a tight cluster represented in red colored nodes. The confidence level was set to 0.4 (medium). PPI enrichment p -value:< 1.0e-16. d-ii Heatmap of the representative genes that have been used in String analysis confirmed to be upregulated in the ROCK2 hi cells. Clustvis webtool based on R-software was used for the analysis. Color key shows the differential expression of the genes

Article Snippet: The primary antibodies used were ROCK1(sc-5560), ROCK2(sc-5561), ROCK2(CST-9029), ROCK2(sc-398519), MRE11(CST-4847), NBS1(CST-3001), RAD50(CST-3427), C-PARP (CST-9541), pH2Ax (Calbiochem DR1017), DNA-PK (CST-4602), β-ACTIN (Sigma, clone AC-74), Tubulin (Sigma N6786), RhoC (CST-D40E4), Histone 3 (CST-D1H2), CDK1 (sc-54), pCDK1(CST-9114), pP53-Ser15 (CST-9284), pAKT-Ser473 (CST-9271) and GAPDH (sc-47724).

Techniques: Irradiation, Migration, Software, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

doi: 10.1186/s13046-019-1385-7

Figure Lengend Snippet: ROCK2’s crosstalk with DNA repair machinery in cervical cancer cells. a Immunoblot of irradiated SiHa cells showing expression levels of DNA repair proteins at various time points indicated, as compared to control at 0 h. b-i Representative immunofluorescence images depict the co-expression of ROCK2 and pH2Ax in CaSki cells at 1 h following irradiation ( n = 3, scale bar = 10 μm). b-ii Representative immunofluorescent images of irradiated CaSki cells showing a decreased expression of pH2Ax in ROCK2 In as compared to IgG In cells ( n = 3, scale bar = 10 μm). b-iii Box plots of the number of pH2Ax foci in IgG In and ROCK2 In cells following irradiation. A median value of 15 foci/cell in IgG In as opposed to 5 foci/cell in the ROCK2 In was observed ( n = 3, * p < 0.01). b-iv Immunoblot analysis demonstrated a reduction in pH2Ax levels at 1 h in irradiated ROCK2 In as compared to IgG In cells ( n = 3). b-v Immunoprecipitation using ROCK2 antibody shows pull down of pH2Ax at 1 h following irradiation treatment. IgG isotype is used as the control ( n = 3). c-i Representative immunofluorescence images showing a decreased expression of RAD50 as compared to IgG In , in irradiated CaSki cells, with ROCK2 In ( n = 3, scale bar = 10 μm). c-ii Immunoblot analysis in irradiated SiHa cells confirming reduction in RAD50 levels upon ROCK2 In ( n = 3). d-i Representative immunofluorescence images showing a decreased expression of MRE11 as compared to IgG In cells, in irradiated CaSki cells, with ROCK2 In ( n = 3, scale bar = 10 μm). d-ii Immunoblot analysis of irradiated SiHa cells also showed a reduction in MRE11 levels upon ROCK2 In ( n = 3)

Article Snippet: The primary antibodies used were ROCK1(sc-5560), ROCK2(sc-5561), ROCK2(CST-9029), ROCK2(sc-398519), MRE11(CST-4847), NBS1(CST-3001), RAD50(CST-3427), C-PARP (CST-9541), pH2Ax (Calbiochem DR1017), DNA-PK (CST-4602), β-ACTIN (Sigma, clone AC-74), Tubulin (Sigma N6786), RhoC (CST-D40E4), Histone 3 (CST-D1H2), CDK1 (sc-54), pCDK1(CST-9114), pP53-Ser15 (CST-9284), pAKT-Ser473 (CST-9271) and GAPDH (sc-47724).

Techniques: Western Blot, Irradiation, Expressing, Immunofluorescence, Immunoprecipitation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

doi: 10.1186/s13046-019-1385-7

Figure Lengend Snippet: ROCK2 hi cells have an enhanced cell survival and are primed for a G2-M transition. a-i Representative histograms of flow cytometry analysis displaying the cell cycle profile of irradiated SiHa cells. The cell cycle plots were obtained after gating based on ROCK2 expression i.e., ROCK2 high expressing (ROCK2 hi ) cells and ROCK2 low expressing (ROCK2 lo ) cells. a-ii Graphical representation of distribution of cells in various phases of cell cycle, showing that the ROCK2 hi cells have a significant percentage of cells in G2/M phase as compared to the ROCK2 lo cells ( n = 3). a-iii Graphical representation of percentage survival of ROCK2 hi cells and ROCK2 lo cells. ROCK2 hi cells shows a better survival ( n = 3, * p < 0.02). b-i Graphical representation of the increased percentages of pAKT-Ser473 and P53-Ser15 in the SiHa cells following radiation treatment as compared to the untreated controls, * p < 0.03; ** p < 0.03 ( n = 3). b-ii Graphical representation of the distribution of pAKT-Ser473 and P53-Ser15 in ROCK2 hi population as compared to ROCK2 lo cells, gated based on ROCK2 expression levels in the irradiated SiHa cells, * p < 0.009; ** p < 0.03 ( n = 3). c-i Immunofluorescence staining showed an upregulated expression of both ROCK2 and CDK1 upon irradiation (scale bar = 10 μm). c-ii Flow cytometric analysis of irradiated SiHa cells showed an increased percentage of cells that co-expressed ROCK2 and CDK1 as compared to the corresponding control. c-iii Graphical representation of the distribution of pCDK1 levels in the ROCK2 hi population as compared to ROCK2 lo , gated based on ROCK2 expression levels in the irradiated SiHa cells, * p < 0.003 ( n = 3)

Article Snippet: The primary antibodies used were ROCK1(sc-5560), ROCK2(sc-5561), ROCK2(CST-9029), ROCK2(sc-398519), MRE11(CST-4847), NBS1(CST-3001), RAD50(CST-3427), C-PARP (CST-9541), pH2Ax (Calbiochem DR1017), DNA-PK (CST-4602), β-ACTIN (Sigma, clone AC-74), Tubulin (Sigma N6786), RhoC (CST-D40E4), Histone 3 (CST-D1H2), CDK1 (sc-54), pCDK1(CST-9114), pP53-Ser15 (CST-9284), pAKT-Ser473 (CST-9271) and GAPDH (sc-47724).

Techniques: Flow Cytometry, Irradiation, Expressing, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

doi: 10.1186/s13046-019-1385-7

Figure Lengend Snippet: ROCK2 is the downstream effector of RhoC in radiation response. a-i An siRNA-based inhibition of RhoC in SiHa cells followed by immunoblotting of the cell extracts showed a reduction in the protein levels of RhoC and ROCK2 ( n = 3). a-ii Immunoblotting the cell extracts of CaSki cells expressing the dominant negative RhoC (CaSki-dnR) showed a reduction in the ROCK2 levels as compared to the cells containing the empty vector (CaSki-N) ( n = 3). b-i Immunoblotting of the cell extracts from SiHa cells with overexpression of RhoC showed an increase in the levels of ROCK2 as compared to the control cells ( n = 3). b-ii Real time PCR showed a 2.4- fold upregulation of ROCK2 mRNA levels in SiHa-R cells as compared to SiHa-N cells ( n = 3, p < 0.05). b-iii Graphical representation of flow cytometric analysis showed a 1.65-fold increase in the percentage of ROCK2 positive cells in the SiHa-R as compared to SiHa-N cells p < 0.05 ( n = 3). c Graphical representation of flow cytometry analysis of SiHa-R cells with ROCK2 In displayed loss of survival advantage post-irradiation as compared to IgG In cells ( n = 3, * p < 0.03). d Immunoprecipitation using RhoC antibody resulted in pulldown of ROCK2 (~ 120 kDa). e Representative images of immunofluorescence-based analysis of xenografts derived from SiHa-N and SiHa-R tumors showing increased expression of ROCK2 in SiHa-R derived sections (scale bar = 50 μm)

Article Snippet: The primary antibodies used were ROCK1(sc-5560), ROCK2(sc-5561), ROCK2(CST-9029), ROCK2(sc-398519), MRE11(CST-4847), NBS1(CST-3001), RAD50(CST-3427), C-PARP (CST-9541), pH2Ax (Calbiochem DR1017), DNA-PK (CST-4602), β-ACTIN (Sigma, clone AC-74), Tubulin (Sigma N6786), RhoC (CST-D40E4), Histone 3 (CST-D1H2), CDK1 (sc-54), pCDK1(CST-9114), pP53-Ser15 (CST-9284), pAKT-Ser473 (CST-9271) and GAPDH (sc-47724).

Techniques: Inhibition, Western Blot, Expressing, Dominant Negative Mutation, Plasmid Preparation, Over Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Irradiation, Immunoprecipitation, Immunofluorescence, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer

doi: 10.1186/s13046-019-1385-7

Figure Lengend Snippet: Evaluation of ROCK2 as a prospective radiosensitizer in vitro using clinical samples and its association with DNA repair markers. a-i Immunofluorescence analysis shows co-expression of both RhoC and ROCK2 in the nuclear compartment of tumor sections (scale bar = 10 μm). a-ii Immunofluorescence analysis shows co-expression of both pH2Ax and ROCK2 in the nuclear compartment of tumor sections (scale bar = 10 μm). a-iii Immunofluorescence analysis shows co-expression of both RAD50 and ROCK2 in the nuclear compartment of tumor sections (scale bar = 10 μm). a-iv Immunofluorescence analysis shows co-expression of both MRE11 and ROCK2 in the nuclear compartment of tumor sections (scale bar = 10 μm). b(i-iii) Scatter plots depict the percentage of cells that showed nuclear expression of either ROCK2 or pH2Ax or both (R2 + pH2Ax). Similarly, cells were analyzed for nuclear expression of ROCK2-RAD50 and ROCK2-MRE11 as represented in the figure. c-i ROCK2 In was performed in cervical cancer biopsy derived tumor cells with a corresponding IgG In control ( n = 15 samples). Graphical representation of n = 11 samples with ROCK2 In resulted in increased cell death as compared to the control, is shown in the figure p < 0.0001. c-ii ROCK2 In performed in cervical cancer biopsy derived tumor cells along with Cisplatin (Cis) at 20 μM followed by irradiation showed an increased cell death. The graphical representation displays an increased cell death upon combinatorial treatment of cisplatin and ROCK2 as opposed to cisplatin alone ( n = 5 samples)

Article Snippet: The primary antibodies used were ROCK1(sc-5560), ROCK2(sc-5561), ROCK2(CST-9029), ROCK2(sc-398519), MRE11(CST-4847), NBS1(CST-3001), RAD50(CST-3427), C-PARP (CST-9541), pH2Ax (Calbiochem DR1017), DNA-PK (CST-4602), β-ACTIN (Sigma, clone AC-74), Tubulin (Sigma N6786), RhoC (CST-D40E4), Histone 3 (CST-D1H2), CDK1 (sc-54), pCDK1(CST-9114), pP53-Ser15 (CST-9284), pAKT-Ser473 (CST-9271) and GAPDH (sc-47724).

Techniques: In Vitro, Immunofluorescence, Expressing, Derivative Assay, Irradiation

Journal: PLoS Biology

Article Title: Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion

doi: 10.1371/journal.pbio.3000154

Figure Lengend Snippet: (A) Western blot analysis of DMSO- and RAP-treated mature cycle 0 PfPDEβ ΔcatHA schizonts, probed with antibodies specific for the phosphorylated generic consensus PKA substrate motif. The blot was reprobed with an anti-HA antibody to monitor disruption of PfPDEβ-HA expression, as well as an antibody to MyoA as a loading control. (B) Changes in phosphorylation and protein abundance between RAP-treated (PfPDEβ-null, KO) and mock-treated (WT) Compound 2–arrested PfPDEβ ΔcatHA schizonts. Left panel: peptide intensity (log 10 ) plotted against log 2 fold change for 5,374 phosphosites, with significantly altered sites (Welch-corrected t test) in dark grey. Seven phosphosites from the PDEβ N-terminal domain (red) and four significantly up-regulated phosphosites in ACβ and CDPK1 (green), as well as MyoA and AMA1 (blue), are highlighted. Right panel: changes in protein abundance, with PfPDEβ, hDHFR, mTOR, and FKBP1A highlighted. (C) Sequence logo showing consensus sequence surrounding phosphosites (position 0) significantly increased in the PfPDEβ-null samples. (D) Motif analysis showing the six motifs most enriched in the PfPDEβ-null samples by 1D annotation analysis (green) and two control kinase motifs (red). CDPK1 and CRK4 motifs used are described in Materials and methods. Data show mean log 2 fold changes. Error bars, SEM. Numbers in parentheses denote the frequency of the occurrence of the motif in phosphosites significantly up-regulated in the PfPDEβ-null/total number of phosphosites with that motif. (E) Presentation of GO terms dysregulated in PfPDEβ-null schizonts. Bars show numbers of phosphosites up- (green) and down-regulated (red) in the PfPDEβ-null schizonts compared with wild type. Light shades denote sites significantly different by Welch t test, and bright colours denote sites >2-fold changed. Hatched bars mark phosphosites with a minimal PKA consensus motif (K/R, x, S/T). The numbers of proteins in each group are indicated on the right. ACβ, adenylyl cyclase β; AMA1, apical membrane antigen-1; CDPK1, calcium-dependent protein kinase 1; CRK4, cdc2-related protein kinase 4; FKBP1A, FK506-binding protein 1A; GO, gene ontology; HA, haemagglutinin; hDHFR, human dihydrofolate reductase; KO, knockout; K/R, lysine/arginine; mTOR, mechanistic target of rapamycin; MyoA, myosin A; PDEβ, phosphodiesterase β; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; RAP, rapamycin; S/T, serine/threonine; WT, wild type; x, any amino acid; 1D, one-dimensional.

Article Snippet: A rabbit monoclonal antibody specifically reacting with phosphorylated PKA substrate consensus motif (R,K/R,X,pS/pT) was purchased from Cell Signaling Technology.

Techniques: Western Blot, Expressing, Sequencing, Binding Assay, Knock-Out

Journal: PLoS Biology

Article Title: Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion

doi: 10.1371/journal.pbio.3000154

Figure Lengend Snippet: (A) The PDE inhibitor BIPPO phenocopies the PfPDEβ-null post-invasion phenotype. Giemsa-stained blood films showing the morphology of PfPDEβ ΔcatHA parasites at different time points in cycle 1, following either mock or RAP treatment of the parasites in cycle 0, compared to mock-treated parasites exposed to the PDE inhibitor BIPPO immediately following invasion. (B) The PKA inhibitor H89, but not the PKG inhibitor Compound 2, rescues BIPPO-treated early ring stages. The plots were generated by microscopic analysis of Giemsa-stained smears from ring stage cultures (18–22 hpi) treated with the indicated compounds. Kinase inhibitors (H89 and C2) were added at 1–5 hpi and BIPPO at 2–6 hpi. Scale bar, 5 μm. Data presented are mean counts (evaluated by three independent researchers) from two independent experiments, with >100 parasites counted per condition. Error bars, SEM. *, significant by unpaired t test ( p -value = 0.0005); n.s., not significant ( p -value = 0.8508). (C) The PDE inhibitor BIPPO induces PKA-dependent phosphorylation in ring stages. Western blot of total protein from ring stages (12–16 hpi) treated for 90 minutes with various inhibitors: BIPPO (2 μM), H89 (30 μM), C2 (2 μM). Lane 1 = no inhibitor control, lane 2 = BIPPO only, lane 3 = BIPPO + H89, lane 4 = BIPPO + C2. The blot was probed with an antibody to phosphorylated PKA substrate motif. The gel was stained for total protein prior to blotting to show equal loading (right panel). BIPPO, 5-Benzyl-3-isopropyl-1 H -pyrazolo[4,3- d ]pyrimidin-7(6 H )-one; hpi, hours post invasion; PDE, phosphodiesterase; PfPDEβ, Plasmodium falciparum phosphodiesterase β; PKA, cAMP-dependent protein kinase; pS/pT, phosphoserine or phosphothreonine; RAP, rapamycin; R/K, arginine or lysine.

Article Snippet: A rabbit monoclonal antibody specifically reacting with phosphorylated PKA substrate consensus motif (R,K/R,X,pS/pT) was purchased from Cell Signaling Technology.

Techniques: Staining, Generated, Western Blot

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation

doi: 10.1152/ajpendo.00189.2016

Figure Lengend Snippet: Reduced levels of serine/threonine phosphorylated proteins in Lrrk1 KO osteoclasts. Osteoclast precursors derived from WT and Lrrk1 KO mice were differentiated in the presence of RANKL and macrophage colony-stimulating factor (M-CSF) for 4 days, and cells were cultured in serum- and RANKL-free medium for 4 h followed by RANKL stimulation for 30 min. Cellular lysates were analyzed by Western blot using anti-phospho-serine/threonine motif antibodies specific to the PKC substrates A and ATM/ATR substrate B, respectively. Expression of β-actin was used as a loading control. Arrows indicate reduced phosphorylated species of cellular proteins in Lrrk1-deficient osteoclasts.

Article Snippet: Anti-Rac1/Cdc42, anti-pS71-RAC1/Cdc42, anti-pSer 473 -Akt, anti-Akt, anti-PAK1/2/3, anti-pSer 144 -PAK1, anti-pS21-PAK1, anti-pThr 423 -PAK1, anti-PKC substrate serine/threonine motif, and anti-ATM/ATR substrate serine/threonine motif antibodies were products of Cell Signaling Technology (Danvers, MA).

Techniques: Derivative Assay, Cell Culture, Western Blot, Expressing