Journal: Endocrinology

Article Title: P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β-catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice.

doi: 10.1210/en.2012-1781

Figure Lengend Snippet: FIG. 1. Insulin activates Pak1 in gcg-expressing cell lines. A, Detection of the expression of group I Paks (Pak1, 68 kDa; Pak2, 61 kDa; Pak3, 65 kDa) in the GLUTag cell line. Approximately 40 g whole-cell lysate proteins were loaded into each lane for Western blotting, with indicated antibody. B, Colocalization of Pak1 and GLP-1 in mouse distal ileum cryptic L cells. A 5-cm region of mouse distal ileum was isolated and prepared for coimmunostaining of Pak1 (brown) and GLP-1 (red). Arrow points to the same crypt region under various magnification views. Bar indicates 200 m (a); 50 m (b); 20 m (c). C–E, Insulin stimulated Pak1 Thr423 phosphorylation in GLUTag (C), STC-1 (D), and mHypoE-20/2 (E) cell lines. All images are representative blots (n 3).

Article Snippet: Phospho- -cat (Ser675), phos- D ow nloaded from https://academ ic.oup.com /endo/article/154/1/77/2423297 by guest on 15 June 2024 pho-Pak1 (Thr423)/Pak2 (Thr402), Pak1, Pak2, Pak3, phospho-GSK3 (Ser21)/ (Ser9), GSK3 , Akt1, and phosphoCREB1 (Ser133) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Expressing, Western Blot, Isolation, Phospho-proteomics

Journal: Endocrinology

Article Title: P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β-catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice.

doi: 10.1210/en.2012-1781

Figure Lengend Snippet: FIG. 1. Insulin activates Pak1 in gcg-expressing cell lines. A, Detection of the expression of group I Paks (Pak1, 68 kDa; Pak2, 61 kDa; Pak3, 65 kDa) in the GLUTag cell line. Approximately 40 g whole-cell lysate proteins were loaded into each lane for Western blotting, with indicated antibody. B, Colocalization of Pak1 and GLP-1 in mouse distal ileum cryptic L cells. A 5-cm region of mouse distal ileum was isolated and prepared for coimmunostaining of Pak1 (brown) and GLP-1 (red). Arrow points to the same crypt region under various magnification views. Bar indicates 200 m (a); 50 m (b); 20 m (c). C–E, Insulin stimulated Pak1 Thr423 phosphorylation in GLUTag (C), STC-1 (D), and mHypoE-20/2 (E) cell lines. All images are representative blots (n 3).

Article Snippet: Phospho- -cat (Ser675), phos- D ow nloaded from https://academ ic.oup.com /endo/article/154/1/77/2423297 by guest on 15 June 2024 pho-Pak1 (Thr423)/Pak2 (Thr402), Pak1, Pak2, Pak3, phospho-GSK3 (Ser21)/ (Ser9), GSK3 , Akt1, and phosphoCREB1 (Ser133) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Expressing, Western Blot, Isolation, Phospho-proteomics

Journal: Endocrinology

Article Title: P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β-catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice.

doi: 10.1210/en.2012-1781

Figure Lengend Snippet: FIG. 1. Insulin activates Pak1 in gcg-expressing cell lines. A, Detection of the expression of group I Paks (Pak1, 68 kDa; Pak2, 61 kDa; Pak3, 65 kDa) in the GLUTag cell line. Approximately 40 g whole-cell lysate proteins were loaded into each lane for Western blotting, with indicated antibody. B, Colocalization of Pak1 and GLP-1 in mouse distal ileum cryptic L cells. A 5-cm region of mouse distal ileum was isolated and prepared for coimmunostaining of Pak1 (brown) and GLP-1 (red). Arrow points to the same crypt region under various magnification views. Bar indicates 200 m (a); 50 m (b); 20 m (c). C–E, Insulin stimulated Pak1 Thr423 phosphorylation in GLUTag (C), STC-1 (D), and mHypoE-20/2 (E) cell lines. All images are representative blots (n 3).

Article Snippet: Phospho- -cat (Ser675), phos- D ow nloaded from https://academ ic.oup.com /endo/article/154/1/77/2423297 by guest on 15 June 2024 pho-Pak1 (Thr423)/Pak2 (Thr402), Pak1, Pak2, Pak3, phospho-GSK3 (Ser21)/ (Ser9), GSK3 , Akt1, and phosphoCREB1 (Ser133) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Expressing, Western Blot, Isolation, Phospho-proteomics

Journal: Endocrinology

Article Title: P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β-catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice.

doi: 10.1210/en.2012-1781

Figure Lengend Snippet: FIG. 2. Insulin-activated gcg promoter and mRNA expression can be attenuated by the Pak inhibitor IPA3. A, Insulin-stimulated Pak1 activation can be attenuated by IPA3 in GLUTag cells. GLUTag cells were serum starved for 16 h, and pretreated with IPA3 (10 M) or dimethylsulfoxide (DMSO) (vehicle) for 1 h, followed by insulin treatment for 10 min. Line indicates noncontiguous lanes on the same blot. B–E, The stimulatory effect of insulin and cAMP on gcg promoter was attenuated by IPA3 pretreatment. GLUTag cells were transfected with 1 g of 2.4kb-gcg-LUC (B and C), G2S-TK-LUC (D), or 302bp-gcg-LUC (E) for 18 h, followed by serum starvation for 16 h. The cells were then pretreated with IPA3 (10 M) or DMSO for 1 h, followed by insulin (100 nM) or F/I (10 M each) treatment for 4 h. LUC activity is presented as fold change against the control DMSO treatment (n 3 with triplicates in each experiment). F and G, Insulin-stimulated gcg mRNA expression was attenuated by IPA3. GLUTag cells were serum starved for 16 h, followed by IPA3 (10 M) or DMSO pretreatment for 1 h. Total RNA was extracted for qRT-PCR (results normalized against 18S) (F) or Northern blotting (representative blot in which the line indicates the noncontiguous lanes on the same blot, n 3) (G). H, Dominant-negative Pak1 (K299R) cotransfection attenuated insulin-stimulated gcg promoter activity. GLUTag cells were cotransfected with Pak1(K299R) and 2.4 kb-gcg-LUC constructs (n 3). *, P 0.05; **, P 0.01; ***, P 0.001.

Article Snippet: Phospho- -cat (Ser675), phos- D ow nloaded from https://academ ic.oup.com /endo/article/154/1/77/2423297 by guest on 15 June 2024 pho-Pak1 (Thr423)/Pak2 (Thr402), Pak1, Pak2, Pak3, phospho-GSK3 (Ser21)/ (Ser9), GSK3 , Akt1, and phosphoCREB1 (Ser133) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Expressing, Activation Assay, Transfection, Activity Assay, Control, Quantitative RT-PCR, Northern Blot, Dominant Negative Mutation, Cotransfection, Construct

Journal: Endocrinology

Article Title: P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β-catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice.

doi: 10.1210/en.2012-1781

Figure Lengend Snippet: FIG. 1. Insulin activates Pak1 in gcg-expressing cell lines. A, Detection of the expression of group I Paks (Pak1, 68 kDa; Pak2, 61 kDa; Pak3, 65 kDa) in the GLUTag cell line. Approximately 40 g whole-cell lysate proteins were loaded into each lane for Western blotting, with indicated antibody. B, Colocalization of Pak1 and GLP-1 in mouse distal ileum cryptic L cells. A 5-cm region of mouse distal ileum was isolated and prepared for coimmunostaining of Pak1 (brown) and GLP-1 (red). Arrow points to the same crypt region under various magnification views. Bar indicates 200 m (a); 50 m (b); 20 m (c). C–E, Insulin stimulated Pak1 Thr423 phosphorylation in GLUTag (C), STC-1 (D), and mHypoE-20/2 (E) cell lines. All images are representative blots (n 3).

Article Snippet: Phospho- -cat (Ser675), phos- D ow nloaded from https://academ ic.oup.com /endo/article/154/1/77/2423297 by guest on 15 June 2024 pho-Pak1 (Thr423)/Pak2 (Thr402), Pak1, Pak2, Pak3, phospho-GSK3 (Ser21)/ (Ser9), GSK3 , Akt1, and phosphoCREB1 (Ser133) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Expressing, Western Blot, Isolation, Phospho-proteomics

Journal: Endocrinology

Article Title: P21-activated protein kinase 1 (Pak1) mediates the cross talk between insulin and β-catenin on proglucagon gene expression and its ablation affects glucose homeostasis in male C57BL/6 mice.

doi: 10.1210/en.2012-1781

Figure Lengend Snippet: FIG. 2. Insulin-activated gcg promoter and mRNA expression can be attenuated by the Pak inhibitor IPA3. A, Insulin-stimulated Pak1 activation can be attenuated by IPA3 in GLUTag cells. GLUTag cells were serum starved for 16 h, and pretreated with IPA3 (10 M) or dimethylsulfoxide (DMSO) (vehicle) for 1 h, followed by insulin treatment for 10 min. Line indicates noncontiguous lanes on the same blot. B–E, The stimulatory effect of insulin and cAMP on gcg promoter was attenuated by IPA3 pretreatment. GLUTag cells were transfected with 1 g of 2.4kb-gcg-LUC (B and C), G2S-TK-LUC (D), or 302bp-gcg-LUC (E) for 18 h, followed by serum starvation for 16 h. The cells were then pretreated with IPA3 (10 M) or DMSO for 1 h, followed by insulin (100 nM) or F/I (10 M each) treatment for 4 h. LUC activity is presented as fold change against the control DMSO treatment (n 3 with triplicates in each experiment). F and G, Insulin-stimulated gcg mRNA expression was attenuated by IPA3. GLUTag cells were serum starved for 16 h, followed by IPA3 (10 M) or DMSO pretreatment for 1 h. Total RNA was extracted for qRT-PCR (results normalized against 18S) (F) or Northern blotting (representative blot in which the line indicates the noncontiguous lanes on the same blot, n 3) (G). H, Dominant-negative Pak1 (K299R) cotransfection attenuated insulin-stimulated gcg promoter activity. GLUTag cells were cotransfected with Pak1(K299R) and 2.4 kb-gcg-LUC constructs (n 3). *, P 0.05; **, P 0.01; ***, P 0.001.

Article Snippet: Phospho- -cat (Ser675), phos- D ow nloaded from https://academ ic.oup.com /endo/article/154/1/77/2423297 by guest on 15 June 2024 pho-Pak1 (Thr423)/Pak2 (Thr402), Pak1, Pak2, Pak3, phospho-GSK3 (Ser21)/ (Ser9), GSK3 , Akt1, and phosphoCREB1 (Ser133) antibodies were purchased from Cell Signaling (Beverly, MA).

Techniques: Expressing, Activation Assay, Transfection, Activity Assay, Control, Quantitative RT-PCR, Northern Blot, Dominant Negative Mutation, Cotransfection, Construct