Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet:

Article Snippet: The following primary antibodies, diluted per the manufacturers instructions, were used for for western blot analysis of downstream signaling: anti-Rac1 (Proteintech 24072–1-AP), GFP (CST 2955S), GAPDH (Proteintech 60004–1-lg), N-Cadherin (CST D4R1H), Vimentin (CST 5741), Cofilin pS3 (ab12866), Cofilin (ab2824), PAK1 (Thr423)/ PAK2 (Thr402) (CS 2601S), PAK1 (CST 2602S), P38 (Thr180/Tyr182) (CST 4511), P38 (CST 9212), MAP3K1 (Thr1400) (Proteintech 28844–1-AP), and MAP3K1 (Proteintech 19970–1-AP).

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Activation Assay, BIA-KA, Mass Spectrometry, Plasmid Preparation, Software, Control, Transfection, Western Blot

Journal: Human Molecular Genetics

Article Title: PAK1 regulates ATXN1 levels providing an opportunity to modify its toxicity in spinocerebellar ataxia type 1

doi: 10.1093/hmg/ddy200

Figure Lengend Snippet: Inhibition of PAK signaling reduces ATXN1 levels in cells and mice. (A) Treatment of mouse primary cerebellar neuron culture with FRAX486 (PAK1/2/3i), an inhibitor of Group I PAKs, results in a decrease of pPak1 (S144) and Atxn1 levels in a concentration-dependent manner (n = 3, one-way ANOVA, **P < 0.01, ****P < 0.0001. Error bars denote SEM). (B) PF03758309, panPAK inhibitor (panPAKi), modulates Atxn1 levels in a concentration-dependent manner in mouse cerebellar neuron culture (n = 3, one-way ANOVA, ***P < 0.001, ****P < 0.0001. Error bars denote the SEM). (C) Combinatorial delivery of MEK (PD0325901, MEKi) and panPAK (PF03758309, panPAKi) inhibitors in mRFP-ATXN1 [82Q]-IRES-YFP Daoy cell line additively reduces mRFP-ATXN1 [82Q] levels (n = 3, one-way ANOVA, *P < 0.05, ****P < 0.0001. Error bars denote the SEM). (D) Intraperitoneal administration of 15 mg/kg panPAK inhibitor every 8 h for 5 days in Atxn1154Q/2Q mice reduce expanded Atxn1 [154Q] as wells as wild type, Atxn1 [2Q] levels in the cerebellum (n = 4, two-way ANOVA, **P < 0.01. Error bars denote the SEM).

Article Snippet: Primary antibody used: anti-ATXN1 [11 750 ( 35 ), 1:5000], anti-pS776 ATXN1 [PN1248 (3), 1:2000], anti-CIC [126 (22), 1:1000], anti-GAPDH (Advanced ImmunoChemical, 1:20 000), anti-PAK1 (Cell Signaling, 1:1000), anti-pPAK1 (Cell Signaling, 1:800), anti-PAK2 (Cell Signaling, 1:1000), anti-pERK1/2 (Cell Signaling, 1:1000), anti-MSK1 (R&D Systems, 1:1000), anti-pMSK1 (Abcam, 1:800), anti-14–3-3ε (Santa Cruz, 1:800), anti-LC3 (MBL, 1:800) and anti-GFP (Genetex, 1:5000).

Techniques: Inhibition, Concentration Assay

Journal: Molecular Biology of the Cell

Article Title: GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids

doi: 10.1091/mbc.E13-12-0743

Figure Lengend Snippet: GRP78 and OxPAPC-induced activation of Rac signaling. HPAECs were treated with nonspecific or HTJ-11–specific siRNA before OxPAPC (10 μg/ml, 15 min) stimulation. (A) Effect of HTJ-1 knockdown on OxPAPC-induced membrane translocation of GRP78 and cortactin. HTJ-1 protein depletion was verified by Western blot. (B) Rac-GTP pull-down assay of control and HTJ-1–depleted HPAEC. (C) Effect of HTJ-1 knockdown on OxPAPC-induced site-specific phosphorylation of Src, PAK1, and cortactin. Autophosphorylation of Src at Tyr-416, indicating its activation, phosphorylation of Rac target PAK1 at Ser-423 and Ser-199, as well as cortactin phosphorylation at Tyr-421 and Tyr-486, reflecting activation of Rac signaling, were detected by immunoblotting. (D) Effect of HPAEC preincubation with GRP78-blocking antibody on OxPAPC-induced site-specific phosphorylation of PAK1 and cortactin. ECs were pretreated with GRP78-blocking antibody (50 μg/ml), vehicle, or control IgG before OxPAPC stimulation (10 μg/ml).

Article Snippet: Antibodies to phosphocortactin and cortactin were from Millipore (Billerica, MA); GRP78, VE-cadherin, calnexin, calreticulin, and HTJ-1 were from Santa Cruz Biotechnology (Santa Cruz, CA); phospho-PAK1, phospho-Src, Src, Fyn, caveolin-1, and VEGFR2 antibodies were from Cell Signaling (Beverly, MA); GRP78 and Rac1 were from BD Transduction Laboratories (San Diego, CA), EO6 monoclonal antibody recognizing oxidized phosphatidyl choline epitope was from Avanti Polar Lipids (Alabaster, AL).

Techniques: Activation Assay, Translocation Assay, Western Blot, Pull Down Assay, Blocking Assay