Journal: Nature cancer

Article Title: Targeting PAK4 to reprogram the vascular microenvironment and improve CAR-T immunotherapy for glioblastoma

doi: 10.1038/s43018-020-00147-8

Figure Lengend Snippet: a, ECs isolated from human GBM tumor (patient #5377) were transduced to express CRISPR targeting PAK4 or a random sequence, followed by transfection with plasmids expressing WT PAK4 or kinase-dead K350M mutant PAK4 or empty vector (EV). Cell lysates were analyzed by immunoblot. b, Purified MEF2D and PAK4 proteins were incubated in kinase buffer, followed by immunoblot analysis. These experiments were repeated independently twice with similar results.

Article Snippet: After transfer, polyvinylidene difluoride membranes were blotted with the following antibodies: anti-FSP-1 (1:1,000; Millipore; 07–2274), anti-α-SMA (1:1,000; Abcam; ab5694), anti-Snail (1:1,000; Cell Signaling Technology; 3879), anti-Slug (1:1,000; Cell Signaling Technology; 9585), anti-PAK4 (1:1,000; Cell Signaling Technology; 3242), anti-PAK1 (1:1,000; Cell Signaling Technology; 2602), anti-ZEB1 (1:1,000; Cell Signaling Technology (3396) and Thermo Fisher Scientific (PA5–28221)), anti-Claudin-1 (1:1,000; Cell Signaling Technology; 13255), anti-Claudin-4 (1:1,000; Invitrogen; 32–9400), anti-Claudin-5 (1:1,000; Thermo Fisher Scientific; 35–2500), anti-Claudin-14 (1:1,000; Invitrogen; PA5–21602), anti-Occludin (1:1,000; Invitrogen; 71–1500), anti-Jam-2 (1:1,000; Invitrogen; PA5–67831), anti-ICAM-1 (1:500; Cell Signaling Technology; 67836), anti-VCAM-1 (1:500; Cell Signaling Technology; 12367), anti-PPAR-γ (1:1,000; Santa Cruz; sc-7196), anti-MEF2D (1:1,000; BD Biosciences; 610774), anti-phospho-MEF2D-Ser 444 (1:1,000; Millipore; SAB4503938), anti-phospho-MEF2D-Ser 180 (1:1,000; Abnova; PAB15921 ), anti-CD31 (1:1,000; Cell Signaling Technology; 3528), anti-LEF1 (1:1,000; Cell Signaling Technology; 2230) and anti-GAPDH (1:5,000; Cell Signaling Technology; 5174).

Techniques: Isolation, CRISPR, Sequencing, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Western Blot, Purification, Incubation

Journal: Nature cancer

Article Title: Targeting PAK4 to reprogram the vascular microenvironment and improve CAR-T immunotherapy for glioblastoma

doi: 10.1038/s43018-020-00147-8

Figure Lengend Snippet: a, ECs isolated from human GBM tumor (patient #5377) were transduced to express CRISPR targeting PAK4 or a random sequence, followed by transfection with plasmids expressing WT PAK4 or kinase-dead K350M mutant PAK4 or empty vector (EV). Cell lysates were analyzed by immunoblot. b, Purified MEF2D and PAK4 proteins were incubated in kinase buffer, followed by immunoblot analysis. These experiments were repeated independently twice with similar results.

Article Snippet: After transfer, polyvinylidene difluoride membranes were blotted with the following antibodies: anti-FSP-1 (1:1,000; Millipore; 07–2274), anti-α-SMA (1:1,000; Abcam; ab5694), anti-Snail (1:1,000; Cell Signaling Technology; 3879), anti-Slug (1:1,000; Cell Signaling Technology; 9585), anti-PAK4 (1:1,000; Cell Signaling Technology; 3242), anti-PAK1 (1:1,000; Cell Signaling Technology; 2602), anti-ZEB1 (1:1,000; Cell Signaling Technology (3396) and Thermo Fisher Scientific (PA5–28221)), anti-Claudin-1 (1:1,000; Cell Signaling Technology; 13255), anti-Claudin-4 (1:1,000; Invitrogen; 32–9400), anti-Claudin-5 (1:1,000; Thermo Fisher Scientific; 35–2500), anti-Claudin-14 (1:1,000; Invitrogen; PA5–21602), anti-Occludin (1:1,000; Invitrogen; 71–1500), anti-Jam-2 (1:1,000; Invitrogen; PA5–67831), anti-ICAM-1 (1:500; Cell Signaling Technology; 67836), anti-VCAM-1 (1:500; Cell Signaling Technology; 12367), anti-PPAR-γ (1:1,000; Santa Cruz; sc-7196), anti-MEF2D (1:1,000; BD Biosciences; 610774), anti-phospho-MEF2D-Ser 444 (1:1,000; Millipore; SAB4503938), anti-phospho-MEF2D-Ser 180 (1:1,000; Abnova; PAB15921 ), anti-CD31 (1:1,000; Cell Signaling Technology; 3528), anti-LEF1 (1:1,000; Cell Signaling Technology; 2230) and anti-GAPDH (1:5,000; Cell Signaling Technology; 5174).

Techniques: Isolation, CRISPR, Sequencing, Transfection, Expressing, Mutagenesis, Plasmid Preparation, Western Blot, Purification, Incubation

Journal: Revista portuguesa de cardiologia

Article Title: Protein kinase D participates in cardiomyocyte hypertrophy by regulating extracellular signal-regulated and myocyte enhancer factor 2D.

doi: 10.1016/j.repc.2020.08.008

Figure Lengend Snippet: Figure 4 The PKC/PKD/ERK5 pathway is involved in angiotensin II (Ang II)-induced MEF2D activation. (A) Cardiomyocytes were stimulated

Article Snippet: Antibodies against PKD, MEF2D, phosphoKD744/748, phospho-PKD916, and phospho-MEF2D were btained from Cell Signaling Technology (Cambridge, MA); ntibodies against PKC , ERK5, and phospho-ERK5 from Cayan Chemicals (Ann Arbor, MI), SDS-polyacrylamide gels rom Pierce (Rockford, IL), and PVDF and proteingel apparaus from Bio-Rad (Hercules, CA).

Techniques: Activation Assay