pak1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc pak1
    Rac1 activation by Hh and regulation of Rac1-mediated Hh via <t>PAK1.</t> (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
    Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pak1 - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "Hedgehog signaling is controlled by Rac1 activity"

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    Journal: Theranostics

    doi: 10.7150/thno.67702

    Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Cell Culture, Transfection, Western Blot, Isolation, Luciferase

    Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Western Blot, Cell Culture, Negative Control, Immunofluorescence, Staining, Immunohistochemistry, Luciferase, shRNA

    Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Cell Culture, Western Blot, Negative Control, Mutagenesis, In Vitro, Kinase Assay, Immunofluorescence, Staining, Knock-Out, Luciferase

    Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Immunohistochemistry, Staining, Western Blot, Immunofluorescence

    pak1  (Cell Signaling Technology Inc)


    Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
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  • 97

    Structured Review

    Cell Signaling Technology Inc pak1
    Rac1 activation by Hh and regulation of Rac1-mediated Hh via <t>PAK1.</t> (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
    Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pak1/product/Cell Signaling Technology Inc
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pak1 - by Bioz Stars, 2023-02
    97/100 stars

    Images

    1) Product Images from "Hedgehog signaling is controlled by Rac1 activity"

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    Journal: Theranostics

    doi: 10.7150/thno.67702

    Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Cell Culture, Transfection, Western Blot, Isolation, Luciferase

    Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Western Blot, Cell Culture, Negative Control, Immunofluorescence, Staining, Immunohistochemistry, Luciferase, shRNA

    Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Cell Culture, Western Blot, Negative Control, Mutagenesis, In Vitro, Kinase Assay, Immunofluorescence, Staining, Knock-Out, Luciferase

    Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Immunohistochemistry, Staining, Western Blot, Immunofluorescence

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    Cell Signaling Technology Inc pak1
    Rac1 activation by Hh and regulation of Rac1-mediated Hh via <t>PAK1.</t> (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
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    1) Product Images from "Hedgehog signaling is controlled by Rac1 activity"

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    Journal: Theranostics

    doi: 10.7150/thno.67702

    Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Cell Culture, Transfection, Western Blot, Isolation, Luciferase

    Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Western Blot, Cell Culture, Negative Control, Immunofluorescence, Staining, Immunohistochemistry, Luciferase, shRNA

    Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Cell Culture, Western Blot, Negative Control, Mutagenesis, In Vitro, Kinase Assay, Immunofluorescence, Staining, Knock-Out, Luciferase

    Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Immunohistochemistry, Staining, Western Blot, Immunofluorescence

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    Cell Signaling Technology Inc anti pak1
    Rac1 activation by Hh and regulation of Rac1-mediated Hh via <t>PAK1.</t> (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
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    1) Product Images from "Hedgehog signaling is controlled by Rac1 activity"

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    Journal: Theranostics

    doi: 10.7150/thno.67702

    Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Cell Culture, Transfection, Western Blot, Isolation, Luciferase

    Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Western Blot, Cell Culture, Negative Control, Immunofluorescence, Staining, Immunohistochemistry, Luciferase, shRNA

    Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Cell Culture, Western Blot, Negative Control, Mutagenesis, In Vitro, Kinase Assay, Immunofluorescence, Staining, Knock-Out, Luciferase

    Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Immunohistochemistry, Staining, Western Blot, Immunofluorescence

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    Cell Signaling Technology Inc anti pak1
    Rac1 activation by Hh and regulation of Rac1-mediated Hh via <t>PAK1.</t> (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
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    1) Product Images from "Hedgehog signaling is controlled by Rac1 activity"

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    Journal: Theranostics

    doi: 10.7150/thno.67702

    Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Cell Culture, Transfection, Western Blot, Isolation, Luciferase

    Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.
    Figure Legend Snippet: Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Western Blot, Cell Culture, Negative Control, Immunofluorescence, Staining, Immunohistochemistry, Luciferase, shRNA

    Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Activation Assay, Transfection, Cell Culture, Western Blot, Negative Control, Mutagenesis, In Vitro, Kinase Assay, Immunofluorescence, Staining, Knock-Out, Luciferase

    Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.
    Figure Legend Snippet: Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Techniques Used: Immunohistochemistry, Staining, Western Blot, Immunofluorescence

    polyclonal antibody to pak1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal antibody to pak1
    Increased <t>PAK1</t> expression and phosphorylation in SW620 cells. (A) Detergent extracts prepared from SW480 and SW620 cells were analyzed by immunoblotting for phosphorylated (p)PAK1, total PAK1 and tubulin. (B) Graph showing the quantification of total PAK1 levels by densitometric analysis of immunoblots. (C) Quantification of pPAK1 levels. The intensity of the upper pPAK1 band (arrow in A) was measured and normalized to the level of tubulin. Values are the average ± S.E.M. of four independent experiments. One asterisk denotes a significant difference (P < 0.05) and two asterisks, a highly significant difference (P < 0.01) from SW480 cells by Student’s t -test.
    Polyclonal Antibody To Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Increased diacylglycerol kinase ζ expression in human metastatic colon cancer cells augments Rho GTPase activity and contributes to enhanced invasion"

    Article Title: Increased diacylglycerol kinase ζ expression in human metastatic colon cancer cells augments Rho GTPase activity and contributes to enhanced invasion

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-208

    Increased PAK1 expression and phosphorylation in SW620 cells. (A) Detergent extracts prepared from SW480 and SW620 cells were analyzed by immunoblotting for phosphorylated (p)PAK1, total PAK1 and tubulin. (B) Graph showing the quantification of total PAK1 levels by densitometric analysis of immunoblots. (C) Quantification of pPAK1 levels. The intensity of the upper pPAK1 band (arrow in A) was measured and normalized to the level of tubulin. Values are the average ± S.E.M. of four independent experiments. One asterisk denotes a significant difference (P < 0.05) and two asterisks, a highly significant difference (P < 0.01) from SW480 cells by Student’s t -test.
    Figure Legend Snippet: Increased PAK1 expression and phosphorylation in SW620 cells. (A) Detergent extracts prepared from SW480 and SW620 cells were analyzed by immunoblotting for phosphorylated (p)PAK1, total PAK1 and tubulin. (B) Graph showing the quantification of total PAK1 levels by densitometric analysis of immunoblots. (C) Quantification of pPAK1 levels. The intensity of the upper pPAK1 band (arrow in A) was measured and normalized to the level of tubulin. Values are the average ± S.E.M. of four independent experiments. One asterisk denotes a significant difference (P < 0.05) and two asterisks, a highly significant difference (P < 0.01) from SW480 cells by Student’s t -test.

    Techniques Used: Expressing, Western Blot

    p pak1 2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p pak1 2
    A, Expression of Cdc42, Rac1 and RhoA in platelet lysates from the wild type and genetically targeted mice was probed by Western blotting of Cdc42, Rac1 and RhoA. Platelets from Cdc42 gene targeted mice as compared to the poly (I∶C) treated matching wild type mice showed a complete lack of Cdc42 GTPase. The expression of Rac1 and RhoA was not altered in Cdc42 −/− platelets. β-actin expression was used as a loading control. B, Platelet counts (Mean ± SEM) in the Cdc42 −/− mice (n = 11) were significantly lower ( p <0.05) than the platelet counts in the Cdc42 +/+ mice (n = 12). C, CRP (0.2 µg/ml) or thrombin (0.1 U/ml) induced phosphorylation <t>of</t> <t>PAK1/2</t> is inhibited in the Cdc42 −/− mice platelets as compared to the platelets from Cdc42 +/+ mice. Phosphorylation of PAK1/2 was analyzed as described in the section.
    P Pak1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Gene Targeting Implicates Cdc42 GTPase in GPVI and Non-GPVI Mediated Platelet Filopodia Formation, Secretion and Aggregation"

    Article Title: Gene Targeting Implicates Cdc42 GTPase in GPVI and Non-GPVI Mediated Platelet Filopodia Formation, Secretion and Aggregation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022117

    A, Expression of Cdc42, Rac1 and RhoA in platelet lysates from the wild type and genetically targeted mice was probed by Western blotting of Cdc42, Rac1 and RhoA. Platelets from Cdc42 gene targeted mice as compared to the poly (I∶C) treated matching wild type mice showed a complete lack of Cdc42 GTPase. The expression of Rac1 and RhoA was not altered in Cdc42 −/− platelets. β-actin expression was used as a loading control. B, Platelet counts (Mean ± SEM) in the Cdc42 −/− mice (n = 11) were significantly lower ( p <0.05) than the platelet counts in the Cdc42 +/+ mice (n = 12). C, CRP (0.2 µg/ml) or thrombin (0.1 U/ml) induced phosphorylation of PAK1/2 is inhibited in the Cdc42 −/− mice platelets as compared to the platelets from Cdc42 +/+ mice. Phosphorylation of PAK1/2 was analyzed as described in the section.
    Figure Legend Snippet: A, Expression of Cdc42, Rac1 and RhoA in platelet lysates from the wild type and genetically targeted mice was probed by Western blotting of Cdc42, Rac1 and RhoA. Platelets from Cdc42 gene targeted mice as compared to the poly (I∶C) treated matching wild type mice showed a complete lack of Cdc42 GTPase. The expression of Rac1 and RhoA was not altered in Cdc42 −/− platelets. β-actin expression was used as a loading control. B, Platelet counts (Mean ± SEM) in the Cdc42 −/− mice (n = 11) were significantly lower ( p <0.05) than the platelet counts in the Cdc42 +/+ mice (n = 12). C, CRP (0.2 µg/ml) or thrombin (0.1 U/ml) induced phosphorylation of PAK1/2 is inhibited in the Cdc42 −/− mice platelets as compared to the platelets from Cdc42 +/+ mice. Phosphorylation of PAK1/2 was analyzed as described in the section.

    Techniques Used: Expressing, Western Blot

    pak1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pak1
    Inhibition of CDC42 prevents NDRG1 loss induced CRC cell filopodial protrusion formation through suppression of <t>PAK1/Cofilin</t> signaling. A) Immunoblotting analysis of the expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. B) Knockdown of CDC42 in HCT116 (left) and RKO (right) cells confirmed with immunoblotting analysis. Pool, combined siCDC42 sequences. C) Expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. D) Confocal images were taken to show immunofluorescence staining of MYO10 (green) and rhodamine-phalloidin (red) accompanied by the cell nucleus (blue) in colorectal cancer cells. Quantification of the MYO10-associated filopodial protrusions density and length is represented as mean ± S.D.; results are representative of 3-5 images from different visual fields, n>50 cells. *P value <0.05, **P value <0.01, ***P < 0.001, relative to the sh-Con/si-Con groups. # P value <0.05, ## P value <0.01, ### P < 0.001, relative to the sh-NDRG1/si-Con groups.
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    Images

    1) Product Images from "NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity"

    Article Title: NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.56694

    Inhibition of CDC42 prevents NDRG1 loss induced CRC cell filopodial protrusion formation through suppression of PAK1/Cofilin signaling. A) Immunoblotting analysis of the expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. B) Knockdown of CDC42 in HCT116 (left) and RKO (right) cells confirmed with immunoblotting analysis. Pool, combined siCDC42 sequences. C) Expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. D) Confocal images were taken to show immunofluorescence staining of MYO10 (green) and rhodamine-phalloidin (red) accompanied by the cell nucleus (blue) in colorectal cancer cells. Quantification of the MYO10-associated filopodial protrusions density and length is represented as mean ± S.D.; results are representative of 3-5 images from different visual fields, n>50 cells. *P value <0.05, **P value <0.01, ***P < 0.001, relative to the sh-Con/si-Con groups. # P value <0.05, ## P value <0.01, ### P < 0.001, relative to the sh-NDRG1/si-Con groups.
    Figure Legend Snippet: Inhibition of CDC42 prevents NDRG1 loss induced CRC cell filopodial protrusion formation through suppression of PAK1/Cofilin signaling. A) Immunoblotting analysis of the expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. B) Knockdown of CDC42 in HCT116 (left) and RKO (right) cells confirmed with immunoblotting analysis. Pool, combined siCDC42 sequences. C) Expression level of the total and phosphorylation form of PAK1 and Cofilin in indicated cell lines. D) Confocal images were taken to show immunofluorescence staining of MYO10 (green) and rhodamine-phalloidin (red) accompanied by the cell nucleus (blue) in colorectal cancer cells. Quantification of the MYO10-associated filopodial protrusions density and length is represented as mean ± S.D.; results are representative of 3-5 images from different visual fields, n>50 cells. *P value <0.05, **P value <0.01, ***P < 0.001, relative to the sh-Con/si-Con groups. # P value <0.05, ## P value <0.01, ### P < 0.001, relative to the sh-NDRG1/si-Con groups.

    Techniques Used: Inhibition, Western Blot, Expressing, Immunofluorescence, Staining

    Schematic diagram for the mechanism of NDRG1's regulation of CDC42/PAK1/Cofilin axis as a switch that modulates actin cytoskeleton rearrangement in human colorectal cancer invasion by stabilizing the RhoGDIα-CDC42 binding.
    Figure Legend Snippet: Schematic diagram for the mechanism of NDRG1's regulation of CDC42/PAK1/Cofilin axis as a switch that modulates actin cytoskeleton rearrangement in human colorectal cancer invasion by stabilizing the RhoGDIα-CDC42 binding.

    Techniques Used: Binding Assay

    anti pak1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti pak1
    (A) HeLa cells were transfected with siRNA directed against RICTOR and then allowed to adhere to plates coated with invasin (β1 integrin-ligand) or Pluronic (non-adhesive control) for 60 min. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and quantifications of AKT pSer473, FOXO1 pThr24, and BAD pSer136 on Pluronic and invasin are given below (mean ± s.e.m.; n = 3; * represents p <0.005 and ** represents p <0.05). (B) MCF7 cells were transfected and treated in the same manner as in (A). (C) HeLa cells transfected with ILK-directed siRNA or non-target siRNA were allowed to adhere to plates coated with Pluronic, collagen type I or invasin. Cells were lysed and analysed as explained above. A representative western blot is shown and the graph to the right shows quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; n = 3). (D) HeLa cells were transfected simultaneously with <t>PAK1-</t> and PAK2-directed siRNAs or non-target siRNA. Adhesion assays were performed on plates coated with Pluronic, invasin or collagen type I. Cell lysates were subjected to SDS-PAGE (10% gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and the graph below presents quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; n = 3). (E) Adhesion assay with MCF7 cells transfected with PAK1- and PAK2-directed siRNAs or non-target siRNA, performed as described in (A).
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    Images

    1) Product Images from "Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival"

    Article Title: Receptor-Specific Mechanisms Regulate Phosphorylation of AKT at Ser473: Role of RICTOR in β1 Integrin-Mediated Cell Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032081

    (A) HeLa cells were transfected with siRNA directed against RICTOR and then allowed to adhere to plates coated with invasin (β1 integrin-ligand) or Pluronic (non-adhesive control) for 60 min. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and quantifications of AKT pSer473, FOXO1 pThr24, and BAD pSer136 on Pluronic and invasin are given below (mean ± s.e.m.; n = 3; * represents p <0.005 and ** represents p <0.05). (B) MCF7 cells were transfected and treated in the same manner as in (A). (C) HeLa cells transfected with ILK-directed siRNA or non-target siRNA were allowed to adhere to plates coated with Pluronic, collagen type I or invasin. Cells were lysed and analysed as explained above. A representative western blot is shown and the graph to the right shows quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; n = 3). (D) HeLa cells were transfected simultaneously with PAK1- and PAK2-directed siRNAs or non-target siRNA. Adhesion assays were performed on plates coated with Pluronic, invasin or collagen type I. Cell lysates were subjected to SDS-PAGE (10% gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and the graph below presents quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; n = 3). (E) Adhesion assay with MCF7 cells transfected with PAK1- and PAK2-directed siRNAs or non-target siRNA, performed as described in (A).
    Figure Legend Snippet: (A) HeLa cells were transfected with siRNA directed against RICTOR and then allowed to adhere to plates coated with invasin (β1 integrin-ligand) or Pluronic (non-adhesive control) for 60 min. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and quantifications of AKT pSer473, FOXO1 pThr24, and BAD pSer136 on Pluronic and invasin are given below (mean ± s.e.m.; n = 3; * represents p <0.005 and ** represents p <0.05). (B) MCF7 cells were transfected and treated in the same manner as in (A). (C) HeLa cells transfected with ILK-directed siRNA or non-target siRNA were allowed to adhere to plates coated with Pluronic, collagen type I or invasin. Cells were lysed and analysed as explained above. A representative western blot is shown and the graph to the right shows quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; n = 3). (D) HeLa cells were transfected simultaneously with PAK1- and PAK2-directed siRNAs or non-target siRNA. Adhesion assays were performed on plates coated with Pluronic, invasin or collagen type I. Cell lysates were subjected to SDS-PAGE (10% gel) followed by western blotting using the different antibodies as indicated. A representative western blot is shown and the graph below presents quantification of AKT pSer473 levels on Pluronic and invasin (mean ± s.e.m.; n = 3). (E) Adhesion assay with MCF7 cells transfected with PAK1- and PAK2-directed siRNAs or non-target siRNA, performed as described in (A).

    Techniques Used: Transfection, SDS Page, Western Blot, Cell Adhesion Assay

    (A) HeLa cells were transfected with RICTOR-directed siRNA or non-target siRNA and then stimulated with LPA (10 µM) for 20 minutes. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) and LPA-induced AKT Ser473 phosphorylation was determined by western blotting. A representative western blot is shown and the graph below provides quantification of AKT pSer473 (mean ± s.e.m.; n = 3). (B) HeLa cells transfected with ILK-directed siRNA and stimulated by LPA were analysed as described above. The graph below shows quantification of AKT pSer473 (mean ± s.e.m.; n = 2). (C) MCF7 cells transfected as indicated were stimulated with LPA (5 µM) for 5 min and analysed as described above. A representative western blot is presented and the graph below shows quantified levels of AKT pSer473 (mean ± s.e.m.; n = 3, * represents p <0.005). (D) MCF7 cells were transfected simultaneously with PAK1 and PAK2 siRNA or with non-target siRNA and then stimulated with LPA (5 µM) for 5 min. A representative western blot is shown. The graph below is a quantification of AKT pSer473 levels (mean ± s.e.m.; n = 3, ** represents p <0.05). (E) HeLa cells were treated with increasing concentrations of the PAK inhibitor IPA3 or with DMSO and then stimulated with LPA (10 µM) for 20 min. The graph below is the quantification of AKT pSer473 levels after LPA-stimulation in cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in LPA-stimulated cells without the inhibitor (mean ± s.e.m.; n = 3, * represents p <0.005).
    Figure Legend Snippet: (A) HeLa cells were transfected with RICTOR-directed siRNA or non-target siRNA and then stimulated with LPA (10 µM) for 20 minutes. Cell lysates were subjected to SDS-PAGE (4–15% gradient gel) and LPA-induced AKT Ser473 phosphorylation was determined by western blotting. A representative western blot is shown and the graph below provides quantification of AKT pSer473 (mean ± s.e.m.; n = 3). (B) HeLa cells transfected with ILK-directed siRNA and stimulated by LPA were analysed as described above. The graph below shows quantification of AKT pSer473 (mean ± s.e.m.; n = 2). (C) MCF7 cells transfected as indicated were stimulated with LPA (5 µM) for 5 min and analysed as described above. A representative western blot is presented and the graph below shows quantified levels of AKT pSer473 (mean ± s.e.m.; n = 3, * represents p <0.005). (D) MCF7 cells were transfected simultaneously with PAK1 and PAK2 siRNA or with non-target siRNA and then stimulated with LPA (5 µM) for 5 min. A representative western blot is shown. The graph below is a quantification of AKT pSer473 levels (mean ± s.e.m.; n = 3, ** represents p <0.05). (E) HeLa cells were treated with increasing concentrations of the PAK inhibitor IPA3 or with DMSO and then stimulated with LPA (10 µM) for 20 min. The graph below is the quantification of AKT pSer473 levels after LPA-stimulation in cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in LPA-stimulated cells without the inhibitor (mean ± s.e.m.; n = 3, * represents p <0.005).

    Techniques Used: Transfection, SDS Page, Western Blot

    (A) PAK1 and PAK2 expression was suppressed in MCF7 cells using siRNAs and the cells were stimulated with 20 ng/ml PDGF-BB for 10 min. A representative western blot is shown and AKT pSer473 is quantified below (mean ± s.e.m.; n = 2, ** represents p <0.05). (B) MCF7 and HeLa cells were transfected as above and stimulated with 20 ng/ml EGF. A representative western blot is shown and below quantification of AKT pSer473 in MCF7 and HeLa cells (mean ± s.e.m.; n = 3) is presented. (C) HeLa cells were treated with PAK inhibitor IPA3 (30 µM) or DMSO as vehicle control and then stimulated with 20 ng/ml PDGF-BB or EGF. A representative western blot is shown. The graph provides quantification of AKT pSer473 levels, after EGF-stimulation of cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in EGF-stimulated cells without the inhibitor (mean ± s.e.m.; n = 3). (D) RICTOR expression was suppressed in HeLa cells using siRNA and the cells were stimulated with EGF (20 ng/ml). Cell lysates were subjected to SDS-PAGE followed by western blotting using antibodies as indicated. A representative blot is shown and quantification of AKT pS473 levels is found below (mean ± s.e.m.; n = 3, ** represents p <0.05). (E) HeLa cells transfected with RICTOR-directed siRNA were stimulated with 20 ng/ml PDGF-BB and analysed as in (D). A representative western blot is shown and the graph below shows quantification of AKT pSer473 (mean ± s.e.m.; n = 2, * represents p <0.005). (F) HeLa cells, transfected with non-target or ILK-directed siRNA, were stimulated with 20 ng/ml EGF and the samples analysed as explained above. The graph shows quantification of AKT pSer473 (mean ± s.e.m.; n = 2).
    Figure Legend Snippet: (A) PAK1 and PAK2 expression was suppressed in MCF7 cells using siRNAs and the cells were stimulated with 20 ng/ml PDGF-BB for 10 min. A representative western blot is shown and AKT pSer473 is quantified below (mean ± s.e.m.; n = 2, ** represents p <0.05). (B) MCF7 and HeLa cells were transfected as above and stimulated with 20 ng/ml EGF. A representative western blot is shown and below quantification of AKT pSer473 in MCF7 and HeLa cells (mean ± s.e.m.; n = 3) is presented. (C) HeLa cells were treated with PAK inhibitor IPA3 (30 µM) or DMSO as vehicle control and then stimulated with 20 ng/ml PDGF-BB or EGF. A representative western blot is shown. The graph provides quantification of AKT pSer473 levels, after EGF-stimulation of cells treated with IPA3 (30 µM) normalised to the pSer473 level of this protein in EGF-stimulated cells without the inhibitor (mean ± s.e.m.; n = 3). (D) RICTOR expression was suppressed in HeLa cells using siRNA and the cells were stimulated with EGF (20 ng/ml). Cell lysates were subjected to SDS-PAGE followed by western blotting using antibodies as indicated. A representative blot is shown and quantification of AKT pS473 levels is found below (mean ± s.e.m.; n = 3, ** represents p <0.05). (E) HeLa cells transfected with RICTOR-directed siRNA were stimulated with 20 ng/ml PDGF-BB and analysed as in (D). A representative western blot is shown and the graph below shows quantification of AKT pSer473 (mean ± s.e.m.; n = 2, * represents p <0.005). (F) HeLa cells, transfected with non-target or ILK-directed siRNA, were stimulated with 20 ng/ml EGF and the samples analysed as explained above. The graph shows quantification of AKT pSer473 (mean ± s.e.m.; n = 2).

    Techniques Used: Expressing, Western Blot, Transfection, SDS Page

    antibodies against pak1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc antibodies against pak1
    Cell proteins were separated using gel electrophoresis and the expression levels of <t>PAK1,</t> PAK2, phospho-PAK (Ser 144/Ser141) and phospho-PAK2 (Ser20) were determined using western-blotting. (A) Lanes 1–8 correspond to the individual cell lines: (1) JURL-MK1, (2) MOLM-7, (3) K562, (4) CML-T1, (5) HL60, (6) Karpas-299, (7) Jurkat, (8) HEL. (B) CML-T1 cells were treated with IPA-3 at the indicated concetrations for 2 h, then lysed and subjected to electrophoresis and western-blotting. (C) Relative expression levels of phospho-PAK2 (Ser141) and PAK2 in cell lines treated for 2 h with IPA-3 at different concentrations. The band intensity from western-blots with anti-PAK antibodies was corrected for small differencies in protein loads using actin bands and the resulting values were expressed as relative to controls (without treatment). JURL-MK1:closed circles, MOLM-7:closed triangles, CML-T1:closed squares, K562:open circles, HL60:open squares, JURKAT:open triangles, Karpas-299:open diamonds, HEL:closed diamonds.
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    Images

    1) Product Images from "Group I PAK Inhibitor IPA-3 Induces Cell Death and Affects Cell Adhesivity to Fibronectin in Human Hematopoietic Cells"

    Article Title: Group I PAK Inhibitor IPA-3 Induces Cell Death and Affects Cell Adhesivity to Fibronectin in Human Hematopoietic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092560

    Cell proteins were separated using gel electrophoresis and the expression levels of PAK1, PAK2, phospho-PAK (Ser 144/Ser141) and phospho-PAK2 (Ser20) were determined using western-blotting. (A) Lanes 1–8 correspond to the individual cell lines: (1) JURL-MK1, (2) MOLM-7, (3) K562, (4) CML-T1, (5) HL60, (6) Karpas-299, (7) Jurkat, (8) HEL. (B) CML-T1 cells were treated with IPA-3 at the indicated concetrations for 2 h, then lysed and subjected to electrophoresis and western-blotting. (C) Relative expression levels of phospho-PAK2 (Ser141) and PAK2 in cell lines treated for 2 h with IPA-3 at different concentrations. The band intensity from western-blots with anti-PAK antibodies was corrected for small differencies in protein loads using actin bands and the resulting values were expressed as relative to controls (without treatment). JURL-MK1:closed circles, MOLM-7:closed triangles, CML-T1:closed squares, K562:open circles, HL60:open squares, JURKAT:open triangles, Karpas-299:open diamonds, HEL:closed diamonds.
    Figure Legend Snippet: Cell proteins were separated using gel electrophoresis and the expression levels of PAK1, PAK2, phospho-PAK (Ser 144/Ser141) and phospho-PAK2 (Ser20) were determined using western-blotting. (A) Lanes 1–8 correspond to the individual cell lines: (1) JURL-MK1, (2) MOLM-7, (3) K562, (4) CML-T1, (5) HL60, (6) Karpas-299, (7) Jurkat, (8) HEL. (B) CML-T1 cells were treated with IPA-3 at the indicated concetrations for 2 h, then lysed and subjected to electrophoresis and western-blotting. (C) Relative expression levels of phospho-PAK2 (Ser141) and PAK2 in cell lines treated for 2 h with IPA-3 at different concentrations. The band intensity from western-blots with anti-PAK antibodies was corrected for small differencies in protein loads using actin bands and the resulting values were expressed as relative to controls (without treatment). JURL-MK1:closed circles, MOLM-7:closed triangles, CML-T1:closed squares, K562:open circles, HL60:open squares, JURKAT:open triangles, Karpas-299:open diamonds, HEL:closed diamonds.

    Techniques Used: Nucleic Acid Electrophoresis, Expressing, Western Blot, Electrophoresis

    Cells were nucleofected with 2 μl esiRNA against PAK1 or PAK2, with the combination of both esiRNAs (PAK1+PAK2) or with non-targeting siRNA (ctrl) and the cell adhesion to fibronectin (ACF- adherent cell fraction) was tested 22 h post nucleofection. The level of PAK1, PAK2 and pPAK2 (Ser141) was assessed at the same time by western-bloting. (A) The experiment with the highest silencing efficacy obtained. The graph of cell adhesivity shows means and standard deviations of sample quadruplicates. (B) Summary of results for all adhesion experiments (N = 7). The adherent cell fraction was expressed as relative to the adhesivity of controls and the resulting values were compared using paired Student’s t-test.
    Figure Legend Snippet: Cells were nucleofected with 2 μl esiRNA against PAK1 or PAK2, with the combination of both esiRNAs (PAK1+PAK2) or with non-targeting siRNA (ctrl) and the cell adhesion to fibronectin (ACF- adherent cell fraction) was tested 22 h post nucleofection. The level of PAK1, PAK2 and pPAK2 (Ser141) was assessed at the same time by western-bloting. (A) The experiment with the highest silencing efficacy obtained. The graph of cell adhesivity shows means and standard deviations of sample quadruplicates. (B) Summary of results for all adhesion experiments (N = 7). The adherent cell fraction was expressed as relative to the adhesivity of controls and the resulting values were compared using paired Student’s t-test.

    Techniques Used: esiRNA, Western Blot

    pak1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pak1
    A) I/NI (Ischemic/Non-Ischemic) plantar perfusion ratio is comparable between <t>PAK1</t> −/− and PAK1 +/+ mice as measured by laser Doppler imaging from day 0, immediately after HLI surgery, and throughout the 21 days after HLI surgery. B) Limb use score was determined as described in and is equivalent between groups, where a higher score is observed within the first 2 weeks after surgery and decreased significantly thereafter, and indicating recovery of limb function. C) Appearance scores of PAK1 +/+ and PAK1 −/− mice were not statistically significant. D) Laser Doppler images obtained immediately following HLI surgery and on day 21 reflect a lack of difference in perfusion between PAK1 +/+ (N = 8) and PAK −/− (N = 11) mice.
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    1) Product Images from "Potential Compensation among Group I PAK Members in Hindlimb Ischemia and Wound Healing"

    Article Title: Potential Compensation among Group I PAK Members in Hindlimb Ischemia and Wound Healing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0112239

    A) I/NI (Ischemic/Non-Ischemic) plantar perfusion ratio is comparable between PAK1 −/− and PAK1 +/+ mice as measured by laser Doppler imaging from day 0, immediately after HLI surgery, and throughout the 21 days after HLI surgery. B) Limb use score was determined as described in and is equivalent between groups, where a higher score is observed within the first 2 weeks after surgery and decreased significantly thereafter, and indicating recovery of limb function. C) Appearance scores of PAK1 +/+ and PAK1 −/− mice were not statistically significant. D) Laser Doppler images obtained immediately following HLI surgery and on day 21 reflect a lack of difference in perfusion between PAK1 +/+ (N = 8) and PAK −/− (N = 11) mice.
    Figure Legend Snippet: A) I/NI (Ischemic/Non-Ischemic) plantar perfusion ratio is comparable between PAK1 −/− and PAK1 +/+ mice as measured by laser Doppler imaging from day 0, immediately after HLI surgery, and throughout the 21 days after HLI surgery. B) Limb use score was determined as described in and is equivalent between groups, where a higher score is observed within the first 2 weeks after surgery and decreased significantly thereafter, and indicating recovery of limb function. C) Appearance scores of PAK1 +/+ and PAK1 −/− mice were not statistically significant. D) Laser Doppler images obtained immediately following HLI surgery and on day 21 reflect a lack of difference in perfusion between PAK1 +/+ (N = 8) and PAK −/− (N = 11) mice.

    Techniques Used: Imaging

    A) I/NI foot perfusion ratio is similar between PAK1+/+ and PAK1−/− mice immediately following surgery, but starts to diverge by days 3 and 7 days (B–C). Worse use and appearance scores in PAK1 −/− mice on days 7 and 14 reflect the overall impaired limb function due to impaired neovascularization. D) Laser Doppler images obtained on day 0 pre- and post-surgery, and on day 21 showing similar perfusion between PAK1 −/− (N = 8) and PAK1 +/+ (N = 8) mice.
    Figure Legend Snippet: A) I/NI foot perfusion ratio is similar between PAK1+/+ and PAK1−/− mice immediately following surgery, but starts to diverge by days 3 and 7 days (B–C). Worse use and appearance scores in PAK1 −/− mice on days 7 and 14 reflect the overall impaired limb function due to impaired neovascularization. D) Laser Doppler images obtained on day 0 pre- and post-surgery, and on day 21 showing similar perfusion between PAK1 −/− (N = 8) and PAK1 +/+ (N = 8) mice.

    Techniques Used:

    A) Western blots showing upregulated PAK2 expression and a trend towards increased phospho-PAK2 in PAK1 −/− mice (lanes represent samples from 3 different mice). We did not observe expression of PAK3 in gastrocnemius tissue; however, abundant PAK3 expression is found in mouse brain tissue as can be seen from a positive control sample in lane 7 of the PAK3 blot. B–C) Densitometry analysis reveals a 2-fold increase in PAK2 expression in PAK1 −/− compared to PAK1 +/+ mice, normalized to GAPDH as a loading control, and a concomitant increase in phospho-PAK2 relative to total PAK2. *indicates p≤0.05 using Student's T-test, n = 6. D) Densitometric analysis of total PAK2 protein expression in non-ischemic muscle did not reveal a change in PAK1 +/+ versus PAK1 −/− mice.
    Figure Legend Snippet: A) Western blots showing upregulated PAK2 expression and a trend towards increased phospho-PAK2 in PAK1 −/− mice (lanes represent samples from 3 different mice). We did not observe expression of PAK3 in gastrocnemius tissue; however, abundant PAK3 expression is found in mouse brain tissue as can be seen from a positive control sample in lane 7 of the PAK3 blot. B–C) Densitometry analysis reveals a 2-fold increase in PAK2 expression in PAK1 −/− compared to PAK1 +/+ mice, normalized to GAPDH as a loading control, and a concomitant increase in phospho-PAK2 relative to total PAK2. *indicates p≤0.05 using Student's T-test, n = 6. D) Densitometric analysis of total PAK2 protein expression in non-ischemic muscle did not reveal a change in PAK1 +/+ versus PAK1 −/− mice.

    Techniques Used: Western Blot, Expressing, Positive Control

    A) A notable activation of ERK1/2 but not AKT is observed in PAK1−/− gastrocnemius tissue one hour after HLI surgery. Western blotting analysis of PAK1 −/− non-ischemic and ischemic gastrocnemius muscle shows enhanced phosphorylation of ERK1/2 on Thr202/Tyr204 compared to PAK1 +/+ tissue, whereas phosphorylation of AKT was unchanged. Lanes are grouped into Non-Ischemic and Ischemic from either PAK1 +/+ and PAK1 −/− mice. B and D) Densitometric analysis of non-ischemic tissue from PAK1 +/+ and PAK1 −/− show an increase in pERK1/2 activation (p = 0.03 and 0.06 for pERK1 and pERK2 respectively, Student's T-test) whereas a difference in pAKT activation is not observed. C and E) In ischemic tissue a trend towards an increase in pERK1 (p = 0.08, Student's T-test) but not pERK2 or pAKT in PAK1 −/− compared to control PAK1 +/+ was observed. A total of 3 PAK1 +/+ and PAK1 −/− mice were analyzed. Blots for pERK1/2 and pAKT were normalized to tERK1/2 and tAKT respectively.
    Figure Legend Snippet: A) A notable activation of ERK1/2 but not AKT is observed in PAK1−/− gastrocnemius tissue one hour after HLI surgery. Western blotting analysis of PAK1 −/− non-ischemic and ischemic gastrocnemius muscle shows enhanced phosphorylation of ERK1/2 on Thr202/Tyr204 compared to PAK1 +/+ tissue, whereas phosphorylation of AKT was unchanged. Lanes are grouped into Non-Ischemic and Ischemic from either PAK1 +/+ and PAK1 −/− mice. B and D) Densitometric analysis of non-ischemic tissue from PAK1 +/+ and PAK1 −/− show an increase in pERK1/2 activation (p = 0.03 and 0.06 for pERK1 and pERK2 respectively, Student's T-test) whereas a difference in pAKT activation is not observed. C and E) In ischemic tissue a trend towards an increase in pERK1 (p = 0.08, Student's T-test) but not pERK2 or pAKT in PAK1 −/− compared to control PAK1 +/+ was observed. A total of 3 PAK1 +/+ and PAK1 −/− mice were analyzed. Blots for pERK1/2 and pAKT were normalized to tERK1/2 and tAKT respectively.

    Techniques Used: Activation Assay, Western Blot

    A) Similar EC sprouting is observed in PAK1 −/− and PAK +/+ aortic rings cultured for 5 days in growth media supplemented with FBS, whereas a notable reduction in sprouting was observed following 30 µM IPA3 treatment. B) Magnification of the selected region showing EC sprouts (white arrows). C) Quantification of EC sprouts shows less EC sprouts in PAK1 +/+ and PAK1 −/− with IPA3 treatment. Data points represent the average values ±SEM from 3–9 rings from 3–4 mice per group; * denotes p<0.05 compared to FBS alone.
    Figure Legend Snippet: A) Similar EC sprouting is observed in PAK1 −/− and PAK +/+ aortic rings cultured for 5 days in growth media supplemented with FBS, whereas a notable reduction in sprouting was observed following 30 µM IPA3 treatment. B) Magnification of the selected region showing EC sprouts (white arrows). C) Quantification of EC sprouts shows less EC sprouts in PAK1 +/+ and PAK1 −/− with IPA3 treatment. Data points represent the average values ±SEM from 3–9 rings from 3–4 mice per group; * denotes p<0.05 compared to FBS alone.

    Techniques Used: Cell Culture

    A) Recovery of 2.0 mm ear punch wounds is represented by (Area day X/ Area day 0 ) ×100. There was a notably larger but not statistically significant wound diameter in PAK1 −/− mice compared to PAK1 +/+ mice on day 7. B) The wound edge was stained with Mason's trichrome and shows a very similar wound closure between the two groups by day 28. n = 3 for both PAK1 +/+ and PAK1 −/− groups.
    Figure Legend Snippet: A) Recovery of 2.0 mm ear punch wounds is represented by (Area day X/ Area day 0 ) ×100. There was a notably larger but not statistically significant wound diameter in PAK1 −/− mice compared to PAK1 +/+ mice on day 7. B) The wound edge was stained with Mason's trichrome and shows a very similar wound closure between the two groups by day 28. n = 3 for both PAK1 +/+ and PAK1 −/− groups.

    Techniques Used: Staining

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    Cell Signaling Technology Inc pak1
    Rac1 activation by Hh and regulation of Rac1-mediated Hh via <t>PAK1.</t> (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
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    97
    Cell Signaling Technology Inc anti pak1
    Rac1 activation by Hh and regulation of Rac1-mediated Hh via <t>PAK1.</t> (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.
    Anti Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc polyclonal antibody to pak1
    Increased <t>PAK1</t> expression and phosphorylation in SW620 cells. (A) Detergent extracts prepared from SW480 and SW620 cells were analyzed by immunoblotting for phosphorylated (p)PAK1, total PAK1 and tubulin. (B) Graph showing the quantification of total PAK1 levels by densitometric analysis of immunoblots. (C) Quantification of pPAK1 levels. The intensity of the upper pPAK1 band (arrow in A) was measured and normalized to the level of tubulin. Values are the average ± S.E.M. of four independent experiments. One asterisk denotes a significant difference (P < 0.05) and two asterisks, a highly significant difference (P < 0.01) from SW480 cells by Student’s t -test.
    Polyclonal Antibody To Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p pak1 2
    A, Expression of Cdc42, Rac1 and RhoA in platelet lysates from the wild type and genetically targeted mice was probed by Western blotting of Cdc42, Rac1 and RhoA. Platelets from Cdc42 gene targeted mice as compared to the poly (I∶C) treated matching wild type mice showed a complete lack of Cdc42 GTPase. The expression of Rac1 and RhoA was not altered in Cdc42 −/− platelets. β-actin expression was used as a loading control. B, Platelet counts (Mean ± SEM) in the Cdc42 −/− mice (n = 11) were significantly lower ( p <0.05) than the platelet counts in the Cdc42 +/+ mice (n = 12). C, CRP (0.2 µg/ml) or thrombin (0.1 U/ml) induced phosphorylation <t>of</t> <t>PAK1/2</t> is inhibited in the Cdc42 −/− mice platelets as compared to the platelets from Cdc42 +/+ mice. Phosphorylation of PAK1/2 was analyzed as described in the section.
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    Cell Signaling Technology Inc antibodies against pak1
    Cell proteins were separated using gel electrophoresis and the expression levels of <t>PAK1,</t> PAK2, phospho-PAK (Ser 144/Ser141) and phospho-PAK2 (Ser20) were determined using western-blotting. (A) Lanes 1–8 correspond to the individual cell lines: (1) JURL-MK1, (2) MOLM-7, (3) K562, (4) CML-T1, (5) HL60, (6) Karpas-299, (7) Jurkat, (8) HEL. (B) CML-T1 cells were treated with IPA-3 at the indicated concetrations for 2 h, then lysed and subjected to electrophoresis and western-blotting. (C) Relative expression levels of phospho-PAK2 (Ser141) and PAK2 in cell lines treated for 2 h with IPA-3 at different concentrations. The band intensity from western-blots with anti-PAK antibodies was corrected for small differencies in protein loads using actin bands and the resulting values were expressed as relative to controls (without treatment). JURL-MK1:closed circles, MOLM-7:closed triangles, CML-T1:closed squares, K562:open circles, HL60:open squares, JURKAT:open triangles, Karpas-299:open diamonds, HEL:closed diamonds.
    Antibodies Against Pak1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Journal: Theranostics

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    doi: 10.7150/thno.67702

    Figure Lengend Snippet: Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Article Snippet: Antibodies for PAK1, phospho-PAK1 and phospho-Ser/Thr were from Cell Signaling Technology; IFT88, KIF3A, SuFu, c-Myc, Gli1, acetylated-αTubulin (Ac-Tub), Vav2, GAPDH and β-actin antibdies were from Santa Cruz Biotechnology; Ptch1 antibody, Smo antibody and phospho-Vav2 antibody were from Abcam; Gli2 antibody and Rac1 antibody were from Bioss (Beijing, China); Rac2 and Rac3 antibodies were purchased from Huabio (Hangzhou, China); Flag-tag and HA-tag antibodies were from Beyotime (Shanghai, China); GST-tag antibody was from Yeason (Shanghai, China), and antibody anti-Arl13b was from Proteintech.

    Techniques: Activation Assay, Immunofluorescence, Staining, Cell Culture, Transfection, Western Blot, Isolation, Luciferase

    Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Journal: Theranostics

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    doi: 10.7150/thno.67702

    Figure Lengend Snippet: Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Article Snippet: Antibodies for PAK1, phospho-PAK1 and phospho-Ser/Thr were from Cell Signaling Technology; IFT88, KIF3A, SuFu, c-Myc, Gli1, acetylated-αTubulin (Ac-Tub), Vav2, GAPDH and β-actin antibdies were from Santa Cruz Biotechnology; Ptch1 antibody, Smo antibody and phospho-Vav2 antibody were from Abcam; Gli2 antibody and Rac1 antibody were from Bioss (Beijing, China); Rac2 and Rac3 antibodies were purchased from Huabio (Hangzhou, China); Flag-tag and HA-tag antibodies were from Beyotime (Shanghai, China); GST-tag antibody was from Yeason (Shanghai, China), and antibody anti-Arl13b was from Proteintech.

    Techniques: Activation Assay, Transfection, Western Blot, Cell Culture, Negative Control, Immunofluorescence, Staining, Immunohistochemistry, Luciferase, shRNA

    Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Journal: Theranostics

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    doi: 10.7150/thno.67702

    Figure Lengend Snippet: Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Article Snippet: Antibodies for PAK1, phospho-PAK1 and phospho-Ser/Thr were from Cell Signaling Technology; IFT88, KIF3A, SuFu, c-Myc, Gli1, acetylated-αTubulin (Ac-Tub), Vav2, GAPDH and β-actin antibdies were from Santa Cruz Biotechnology; Ptch1 antibody, Smo antibody and phospho-Vav2 antibody were from Abcam; Gli2 antibody and Rac1 antibody were from Bioss (Beijing, China); Rac2 and Rac3 antibodies were purchased from Huabio (Hangzhou, China); Flag-tag and HA-tag antibodies were from Beyotime (Shanghai, China); GST-tag antibody was from Yeason (Shanghai, China), and antibody anti-Arl13b was from Proteintech.

    Techniques: Activation Assay, Transfection, Cell Culture, Western Blot, Negative Control, Mutagenesis, In Vitro, Kinase Assay, Immunofluorescence, Staining, Knock-Out, Luciferase

    Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Journal: Theranostics

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    doi: 10.7150/thno.67702

    Figure Lengend Snippet: Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Article Snippet: Antibodies for PAK1, phospho-PAK1 and phospho-Ser/Thr were from Cell Signaling Technology; IFT88, KIF3A, SuFu, c-Myc, Gli1, acetylated-αTubulin (Ac-Tub), Vav2, GAPDH and β-actin antibdies were from Santa Cruz Biotechnology; Ptch1 antibody, Smo antibody and phospho-Vav2 antibody were from Abcam; Gli2 antibody and Rac1 antibody were from Bioss (Beijing, China); Rac2 and Rac3 antibodies were purchased from Huabio (Hangzhou, China); Flag-tag and HA-tag antibodies were from Beyotime (Shanghai, China); GST-tag antibody was from Yeason (Shanghai, China), and antibody anti-Arl13b was from Proteintech.

    Techniques: Immunohistochemistry, Staining, Western Blot, Immunofluorescence

    Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Journal: Theranostics

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    doi: 10.7150/thno.67702

    Figure Lengend Snippet: Rac1 activation by Hh and regulation of Rac1-mediated Hh via PAK1. (A) Immunofluorescence staining for Smo in MEFs cultured with or without N-Shh at 100 ng/ml for 48 h and NSC23766 (NSC) at 10 μg/ml for 24 h. Primary cilia were indicated by Ac-Tub staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (B,C) Rac1 activation assays in C3H10T1/2 cells transfected with caSmo for 24 h (B) or cultured with SAG at 50 nM for 24 h (C). (D) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (B) and (C). (E,F) Rac1 activation assays in C3H10T1/2 cells transfected with siSmo (E) for 72 h or cultured with Cyclopamine (Cyc., F) at 5 μM for 24 h. (G) Quantification via densitometry (n=3) and statistical analysis of GTP-Rac1 bands of (E) and (F). (H) Immunoblotting analyses of phospho-PAK1 (pPAK1) and PAK1 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 24 h. (I) Immunoblotting analyses of pPAK1 and PAK1 in cerebellum tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Quantification via densitometry (n=3) and statistical analysis of pPAK1 bands of (H) and (I). (K) Immunoblotting analyses of pPAK1 and PAK1 in C3H10T1/2 cells transfected with or without daRac1 for 24 h and cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (L) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs from Shh +/- and Shh -/- embryos. (M) Immunoblotting analyses of pPAK1 and PAK1 in isolated MEFs cultured with or without Cyclopamine (Cyc.) at 5 μM for 24 h. (N) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with the daRac1 and cultured with or without N-Shh at 100 ng/ml or IPA-3 at 1 μM for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (O) C3H10T1/2 cells were cultured with or without IPA-3 at 1 μM for 24 h. Total cell lysates (Input) and anti-SuFu immunoprecipitates (IP, SuFu Ab, +) from total cell lysates were analyzed by immunoblotting with anti-SuFu and anti-Gli1 antibodies. Protein abundance normalized to GAPDH, respectively. **, ## p < 0.01; error bar, SD.

    Article Snippet: Tissues were incubated overnight at 4 °C with the following primary antibodies: anti-IFT88 (sc-84318, Santa Cruz), anti-Gli1 (ab49314, abcam), anti-Ptch1 (ab53715, abcam), anti-KIF3A (sc-376680, Santa Cruz), anti-pVav2 (ab86695, abcam), anti-PAK1 (#2602, Cell Signaling Technology) and anti-pPAK1 (#2601, Cell Signaling Technology).

    Techniques: Activation Assay, Immunofluorescence, Staining, Cell Culture, Transfection, Western Blot, Isolation, Luciferase

    Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Journal: Theranostics

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    doi: 10.7150/thno.67702

    Figure Lengend Snippet: Rac1 activation by Hh via Vav2. (A) C3H10T1/2 cells were transiently transfected with the indicated plasmids. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-Smo antibodies. (B) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Vav2 immunoprecipitates (IP, Vav2 Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Vav2 and anti-Smo antibodies. IgG was used as a negative control for IP. (C) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-Vav2 antibodies. IgG was used as a negative control for IP. (D) C3H10T1/2 cells were cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates (Input) and anti-Smo immunoprecipitates (IP, Smo Ab, +) from total cell lysates were analyzed by immunoblotting with anti-Smo and anti-phospho-Vav2 (pVav2) antibodies. IgG was used as a negative control for IP. (E) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without N-Shh at 100 ng/ml for 0, 6 or 12 h. (F) Immunoblotting analyses of pVav2 and Vav2 in C3H10T1/2 cells cultured with or without SAG at 50 nM for 0, 3 or 6 h. (G) Immunofluorescence staining for pVav2 in MEFs with or without N-Shh at 100 ng/ml for 24 h. Primary cilia were indicated by Arl13b staining. Nuclei were counterstained by DAPI. Bar, 20 μm. (H) Hematoxylin-eosin (H&E) staining and immunohistochemistry staining for pVav2 in cerebella slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (I) Immunoblotting analyses of pVav2 and Vav2 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (J) Immunohistochemistry staining for pVav2 in human clinical sample slides of non-Wnt/Shh-MB and Shh-MB. (K) Rac1 activation assays and immunoblotting analyses for Gli1, pPAK1 as well as PAK1 in C3H10T1/2 cells transfected with or without Myc-Vav2 for 24 h. (L) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 and cultured with or without N-Shh at 100 ng/ml for 24 h. Total cell lysates were subjected to luciferase assay. N=6. (M) C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with Vav2 shRNA and cultured with or without N-Shh at 100 ng/ml. Total cell lysates were subjected to luciferase assay. N=6. Protein abundance normalized to GAPDH, respectively. * p < 0.05; **, ## p < 0.01; error bar, SD.

    Article Snippet: Tissues were incubated overnight at 4 °C with the following primary antibodies: anti-IFT88 (sc-84318, Santa Cruz), anti-Gli1 (ab49314, abcam), anti-Ptch1 (ab53715, abcam), anti-KIF3A (sc-376680, Santa Cruz), anti-pVav2 (ab86695, abcam), anti-PAK1 (#2602, Cell Signaling Technology) and anti-pPAK1 (#2601, Cell Signaling Technology).

    Techniques: Activation Assay, Transfection, Western Blot, Cell Culture, Negative Control, Immunofluorescence, Staining, Immunohistochemistry, Luciferase, shRNA

    Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Journal: Theranostics

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    doi: 10.7150/thno.67702

    Figure Lengend Snippet: Phosphorylated-KIF3A by Rac1 activation binds to and stabilizes IFT88 protein. (A) C3H10T1/2 cells were transfected with or without daRac1 and cultured with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (B) C3H10T1/2 cells were cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and with MG132 at 10 μM. Total cell lysates (Input) and anti-KIF3A immunoprecipitates (IP, KIF3A Ab, +) from total cell lysates were analyzed by immunoblotting with anti-IFT88 and anti-KIF3A antibodies. IgG was used as a negative control for IP. (C) Immunoblotting analyses of IFT88 in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h and transfected with or without KIF3A for 24 h and treated for different time periods with CHX. (D) Immunoblotting analyses of phospho-KIF3A (pKIF3A, pSer/Thr) and KIF3A in C3H10T1/2 cells transfected with or without daRac1 for 24 h. (E) Immunoblotting analyses of pKIF3A and KIF3A in C3H10T1/2 cells cultured with or without NSC23766 (NSC) at 10 μg/ml for 24 h. (F) Conserved phosphorylation sites in KIF3A by PAK1 among species. (G) C3H10T1/2 cells were transfected with or without wild-type Flag-KIF3A (WT) or Flag-KIF3A mutant (S689A, T694A, S698A, 3Mut) and cultured for 48 h. Total cell lysates (Input) and anti-Flag immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Flag and anti-PAK1 antibodies. (H) C3H10T1/2 cells were transfected with the indicated plasmids and cultured with NSC23766 at 10 μg/ml for 48 h. Total cell lysates (Input) and anti-Myc immunoprecipitates (IP) from total cell lysates were analyzed by immunoblotting with anti-Myc and anti-HA antibodies. (I) An in vitro kinase assay of GST-KIF3A and PAK1* in the presence or absence of ATP. Phospho-Ser/Thr (pS/T) was analysed by western blot. (J) Immunofluorescence staining for IFT88 in Kif3a -knockout C3H10T1/2 cells transfected with KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with NSC23766 at 10 μg/ml for 48 h. Primary cilia were indicated by Ac-Tub staining. Bar, 50 μm. (K) Kif3a -knockout C3H10T1/2 cells were transiently transfected with a Gli luciferase reporter together with wild type KIF3A (WT) or KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. Total cell lysates were subjected to luciferase assay. N=6. (L) Kif3a -knockout C3H10T1/2 cells were transiently transfected with KIF3A mutant (2Mut, S689E + T694E) and cultured with or without NSC23766 (NSC) at 10 μg/ml for 48 h. mRNA levels of Gli1 and Ptch1 were analyzed. N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Article Snippet: Tissues were incubated overnight at 4 °C with the following primary antibodies: anti-IFT88 (sc-84318, Santa Cruz), anti-Gli1 (ab49314, abcam), anti-Ptch1 (ab53715, abcam), anti-KIF3A (sc-376680, Santa Cruz), anti-pVav2 (ab86695, abcam), anti-PAK1 (#2602, Cell Signaling Technology) and anti-pPAK1 (#2601, Cell Signaling Technology).

    Techniques: Activation Assay, Transfection, Cell Culture, Western Blot, Negative Control, Mutagenesis, In Vitro, Kinase Assay, Immunofluorescence, Staining, Knock-Out, Luciferase

    Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Journal: Theranostics

    Article Title: Hedgehog signaling is controlled by Rac1 activity

    doi: 10.7150/thno.67702

    Figure Lengend Snippet: Involvement of Rac1 in Shh-MB and limb bud development. (A) Immunohistochemistry staining for PAK1, pPAK1, IFT88, KIF3A, Gli1 and Ptch1 in cerebella tissue slides of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (B) Immunoblotting analyses of PAK1, pPAK1, IFT88, KIF3A, pKIF3A, Gli1 and Ptch1 in cerebella tissues of GFAP-Cre;SmoM2 +/- and SmoM2 +/- mice. (C) Skeletal preparations of the Prx1-Cre;Rac1 f/f and Rac1 f/f mice and their fore- (FL) and hindlimbs (HL) at postnatal day 0 (P0). (D) Immunofluorescence staining for PAK1, pPAK1, IFT88 and KIF3A in limb buds of Prx1-Cre;Rac1 f/f and Rac1 f/f mouse embryos at E10.5. Nuclei were counterstained by DAPI. Bar, 5 μm. (E) Immunofluorescence staining for Gli1 in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . Cytoskeletons were stained by β-actin. Nuclei were counterstained by DAPI. Bar, 20 μm. (F) mRNA levels of Gli1 (left) and Ptch1 (right) in primary mouse embryonic limb bud fibroblasts of Prx1-Cre;Rac1 f/f and Rac1 f/f . N=6. RNA and protein abundance normalized to GAPDH, respectively. ** p < 0.01; error bar, SD.

    Article Snippet: Tissues were incubated overnight at 4 °C with the following primary antibodies: anti-IFT88 (sc-84318, Santa Cruz), anti-Gli1 (ab49314, abcam), anti-Ptch1 (ab53715, abcam), anti-KIF3A (sc-376680, Santa Cruz), anti-pVav2 (ab86695, abcam), anti-PAK1 (#2602, Cell Signaling Technology) and anti-pPAK1 (#2601, Cell Signaling Technology).

    Techniques: Immunohistochemistry, Staining, Western Blot, Immunofluorescence

    Increased PAK1 expression and phosphorylation in SW620 cells. (A) Detergent extracts prepared from SW480 and SW620 cells were analyzed by immunoblotting for phosphorylated (p)PAK1, total PAK1 and tubulin. (B) Graph showing the quantification of total PAK1 levels by densitometric analysis of immunoblots. (C) Quantification of pPAK1 levels. The intensity of the upper pPAK1 band (arrow in A) was measured and normalized to the level of tubulin. Values are the average ± S.E.M. of four independent experiments. One asterisk denotes a significant difference (P < 0.05) and two asterisks, a highly significant difference (P < 0.01) from SW480 cells by Student’s t -test.

    Journal: BMC Cancer

    Article Title: Increased diacylglycerol kinase ζ expression in human metastatic colon cancer cells augments Rho GTPase activity and contributes to enhanced invasion

    doi: 10.1186/1471-2407-14-208

    Figure Lengend Snippet: Increased PAK1 expression and phosphorylation in SW620 cells. (A) Detergent extracts prepared from SW480 and SW620 cells were analyzed by immunoblotting for phosphorylated (p)PAK1, total PAK1 and tubulin. (B) Graph showing the quantification of total PAK1 levels by densitometric analysis of immunoblots. (C) Quantification of pPAK1 levels. The intensity of the upper pPAK1 band (arrow in A) was measured and normalized to the level of tubulin. Values are the average ± S.E.M. of four independent experiments. One asterisk denotes a significant difference (P < 0.05) and two asterisks, a highly significant difference (P < 0.01) from SW480 cells by Student’s t -test.

    Article Snippet: A polyclonal antibody to PAK1 (Catalogue number: 2602) was purchased from Cell Signaling Technologies (Danvers, MA).

    Techniques: Expressing, Western Blot

    A, Expression of Cdc42, Rac1 and RhoA in platelet lysates from the wild type and genetically targeted mice was probed by Western blotting of Cdc42, Rac1 and RhoA. Platelets from Cdc42 gene targeted mice as compared to the poly (I∶C) treated matching wild type mice showed a complete lack of Cdc42 GTPase. The expression of Rac1 and RhoA was not altered in Cdc42 −/− platelets. β-actin expression was used as a loading control. B, Platelet counts (Mean ± SEM) in the Cdc42 −/− mice (n = 11) were significantly lower ( p <0.05) than the platelet counts in the Cdc42 +/+ mice (n = 12). C, CRP (0.2 µg/ml) or thrombin (0.1 U/ml) induced phosphorylation of PAK1/2 is inhibited in the Cdc42 −/− mice platelets as compared to the platelets from Cdc42 +/+ mice. Phosphorylation of PAK1/2 was analyzed as described in the section.

    Journal: PLoS ONE

    Article Title: Gene Targeting Implicates Cdc42 GTPase in GPVI and Non-GPVI Mediated Platelet Filopodia Formation, Secretion and Aggregation

    doi: 10.1371/journal.pone.0022117

    Figure Lengend Snippet: A, Expression of Cdc42, Rac1 and RhoA in platelet lysates from the wild type and genetically targeted mice was probed by Western blotting of Cdc42, Rac1 and RhoA. Platelets from Cdc42 gene targeted mice as compared to the poly (I∶C) treated matching wild type mice showed a complete lack of Cdc42 GTPase. The expression of Rac1 and RhoA was not altered in Cdc42 −/− platelets. β-actin expression was used as a loading control. B, Platelet counts (Mean ± SEM) in the Cdc42 −/− mice (n = 11) were significantly lower ( p <0.05) than the platelet counts in the Cdc42 +/+ mice (n = 12). C, CRP (0.2 µg/ml) or thrombin (0.1 U/ml) induced phosphorylation of PAK1/2 is inhibited in the Cdc42 −/− mice platelets as compared to the platelets from Cdc42 +/+ mice. Phosphorylation of PAK1/2 was analyzed as described in the section.

    Article Snippet: Anti-Cdc42 antibody was purchased from BD Biosciences and anti-PAK and p-PAK1/2, Akt and p-Akt antibodies were obtained from Cell Signaling.

    Techniques: Expressing, Western Blot

    Cell proteins were separated using gel electrophoresis and the expression levels of PAK1, PAK2, phospho-PAK (Ser 144/Ser141) and phospho-PAK2 (Ser20) were determined using western-blotting. (A) Lanes 1–8 correspond to the individual cell lines: (1) JURL-MK1, (2) MOLM-7, (3) K562, (4) CML-T1, (5) HL60, (6) Karpas-299, (7) Jurkat, (8) HEL. (B) CML-T1 cells were treated with IPA-3 at the indicated concetrations for 2 h, then lysed and subjected to electrophoresis and western-blotting. (C) Relative expression levels of phospho-PAK2 (Ser141) and PAK2 in cell lines treated for 2 h with IPA-3 at different concentrations. The band intensity from western-blots with anti-PAK antibodies was corrected for small differencies in protein loads using actin bands and the resulting values were expressed as relative to controls (without treatment). JURL-MK1:closed circles, MOLM-7:closed triangles, CML-T1:closed squares, K562:open circles, HL60:open squares, JURKAT:open triangles, Karpas-299:open diamonds, HEL:closed diamonds.

    Journal: PLoS ONE

    Article Title: Group I PAK Inhibitor IPA-3 Induces Cell Death and Affects Cell Adhesivity to Fibronectin in Human Hematopoietic Cells

    doi: 10.1371/journal.pone.0092560

    Figure Lengend Snippet: Cell proteins were separated using gel electrophoresis and the expression levels of PAK1, PAK2, phospho-PAK (Ser 144/Ser141) and phospho-PAK2 (Ser20) were determined using western-blotting. (A) Lanes 1–8 correspond to the individual cell lines: (1) JURL-MK1, (2) MOLM-7, (3) K562, (4) CML-T1, (5) HL60, (6) Karpas-299, (7) Jurkat, (8) HEL. (B) CML-T1 cells were treated with IPA-3 at the indicated concetrations for 2 h, then lysed and subjected to electrophoresis and western-blotting. (C) Relative expression levels of phospho-PAK2 (Ser141) and PAK2 in cell lines treated for 2 h with IPA-3 at different concentrations. The band intensity from western-blots with anti-PAK antibodies was corrected for small differencies in protein loads using actin bands and the resulting values were expressed as relative to controls (without treatment). JURL-MK1:closed circles, MOLM-7:closed triangles, CML-T1:closed squares, K562:open circles, HL60:open squares, JURKAT:open triangles, Karpas-299:open diamonds, HEL:closed diamonds.

    Article Snippet: Antibodies against PAK1 (#2602), PAK2 (#2615), PAK3 (#2609), PAK1/2/3 (#2604) and pPAK2(Ser20) (#2607) were from Cell Signaling, the antibody against pPAK(Ser141/144) was from Abcam (ab5247) or from Cell Signaling (#2606), anti pSer3-cofilin from Sigma (C8992), anti-cofilin (sc-33779) from Santa Cruz, anti-pSer16-stathmin (#3353) from Cell Signaling and anti-stathmin1 (ab11269) from Abcam.

    Techniques: Nucleic Acid Electrophoresis, Expressing, Western Blot, Electrophoresis

    Cells were nucleofected with 2 μl esiRNA against PAK1 or PAK2, with the combination of both esiRNAs (PAK1+PAK2) or with non-targeting siRNA (ctrl) and the cell adhesion to fibronectin (ACF- adherent cell fraction) was tested 22 h post nucleofection. The level of PAK1, PAK2 and pPAK2 (Ser141) was assessed at the same time by western-bloting. (A) The experiment with the highest silencing efficacy obtained. The graph of cell adhesivity shows means and standard deviations of sample quadruplicates. (B) Summary of results for all adhesion experiments (N = 7). The adherent cell fraction was expressed as relative to the adhesivity of controls and the resulting values were compared using paired Student’s t-test.

    Journal: PLoS ONE

    Article Title: Group I PAK Inhibitor IPA-3 Induces Cell Death and Affects Cell Adhesivity to Fibronectin in Human Hematopoietic Cells

    doi: 10.1371/journal.pone.0092560

    Figure Lengend Snippet: Cells were nucleofected with 2 μl esiRNA against PAK1 or PAK2, with the combination of both esiRNAs (PAK1+PAK2) or with non-targeting siRNA (ctrl) and the cell adhesion to fibronectin (ACF- adherent cell fraction) was tested 22 h post nucleofection. The level of PAK1, PAK2 and pPAK2 (Ser141) was assessed at the same time by western-bloting. (A) The experiment with the highest silencing efficacy obtained. The graph of cell adhesivity shows means and standard deviations of sample quadruplicates. (B) Summary of results for all adhesion experiments (N = 7). The adherent cell fraction was expressed as relative to the adhesivity of controls and the resulting values were compared using paired Student’s t-test.

    Article Snippet: Antibodies against PAK1 (#2602), PAK2 (#2615), PAK3 (#2609), PAK1/2/3 (#2604) and pPAK2(Ser20) (#2607) were from Cell Signaling, the antibody against pPAK(Ser141/144) was from Abcam (ab5247) or from Cell Signaling (#2606), anti pSer3-cofilin from Sigma (C8992), anti-cofilin (sc-33779) from Santa Cruz, anti-pSer16-stathmin (#3353) from Cell Signaling and anti-stathmin1 (ab11269) from Abcam.

    Techniques: esiRNA, Western Blot