Journal: Oncogene

Article Title: Suppression of RAC1 -driven malignant melanoma by Group A PAK inhibitors

doi: 10.1038/onc.2017.400

Figure Lengend Snippet: One-cell embryos were injected with mRNA encoding the indicated genes. A) RNA expression of BRAF V600E , KRAS 4A G12V and Rac1 P29S produce phenotypes similar to Rasopathies at 12hpf in zebrafish embryos. B) Overexpression of proteins increases the activity of p-Erk and p-Pak 1/2; β-actin was used as a loading control. Transgenic fish were injected with Rac1 mRNA, C) and D) Tg (CMLC:GFP) embryos injected with Rac1 P29S were treated with 1µM inhibitors of RAC1 (NSC23766), BRAF (Vemurafenib), PAK1 (Frax-1036) or MEK (PD325901) at 72 hpf. Phase contrast and GFP images are shown in panel C, and quantitated in panel D. Embryo phenotypes were scored as mild abnormal if elongated (body axis ratio >1.1 but <1.3) and/or small or absent eye development was noted, and abnormal if boxy axis ration was >1.3 and/or pericardial edema was also noted.

Article Snippet: Primary antibodies used in this study were: anti-phospho-PAK1 (pSer144) (#2606), -total PAK1 (#2602), -phospho-MEK1/2 (pSer217/221) (#9121), -total MEK1/2 (#9122), -phospho-ERK1/2 (pThr202/pTyr204) (#9101), -total ERK1/2 (#9102), -GPDH (#4138), and -β-actin (#13E5) from Cell Signaling Technology, and anti-RAC1 from Millipore (#07-1464).

Techniques: Injection, RNA Expression, Over Expression, Activity Assay, Control, Transgenic Assay

Journal: Oncogene

Article Title: Suppression of RAC1 -driven malignant melanoma by Group A PAK inhibitors

doi: 10.1038/onc.2017.400

Figure Lengend Snippet: Cell Culture Wound Closure Assay was developed in 6-well confluent plates. After the scratch the different cell lines were treated with 100 nM BRAF inhibitor (vemurafenib), Pak1 inhibitor (Frax-1036), or MEK inhibitor (PD325901). Snapshot images in an inverted microscope were taken after 24h. Migration was quantified by measuring the size of the cell-free area. % migration for each cell line was measured against control (untreated cells), with the latter defined as 100% migration for each cell line. Where significant, p values are shown comparing responses of a RAC1-mutant cell line (YUHEF) to a BRAF-mutant cell line (501mel).

Article Snippet: Primary antibodies used in this study were: anti-phospho-PAK1 (pSer144) (#2606), -total PAK1 (#2602), -phospho-MEK1/2 (pSer217/221) (#9121), -total MEK1/2 (#9122), -phospho-ERK1/2 (pThr202/pTyr204) (#9101), -total ERK1/2 (#9102), -GPDH (#4138), and -β-actin (#13E5) from Cell Signaling Technology, and anti-RAC1 from Millipore (#07-1464).

Techniques: Cell Culture, Wound Closure Assay, Inverted Microscopy, Migration, Control, Mutagenesis

Journal: Physiological Reports

Article Title: Nox2 signaling and muscle fiber remodeling are attenuated by losartan administration during skeletal muscle unloading

doi: 10.14814/phy2.14606

Figure Lengend Snippet: Nuclear content of the atrophy‐inducing transcription factor p53 is dramatically increased following 7 days of hindlimb unloading, even in losartan‐treated animals. Western blot detection of p53 in the nuclear fraction of soleus muscles. Ponceau S Red (PR) stain (~42 kDa mark) used as a loading control for western blotting quantification. Values are presented as fold control of mean ± SEM . Values sharing the same letter are not significantly different ( p < .05): a indicates different from CON; c indicates different from HUL; d indicates different from CONL

Article Snippet: Following blocking, membranes were incubated overnight (4°C) in blocking buffer with the appropriate primary antibody: anti‐nNOS (1:750, Life Technologies, cat # 61–700), anti‐gp91phox (1:1,000, BD Biosciences, cat # 611415), anti‐p67phox (1:2,500, BD Biosciences, cat # 610913), anti‐p47phox (1:750, BD Biosciences, cat # 610355), anti‐Rac‐1 (1:500, EMD Millipore, cat # 07‐1464), anti‐FoxO3a (1:750, Cell Signaling Technology, cat # 12829), and anti‐p53 (1:750, Cell Signaling Technology, cat # 2527).

Techniques: Western Blot, Muscles, Staining, Control