Journal: Frontiers in cellular neuroscience

Article Title: CB 1 Cannabinoid Receptors Stimulate Gβγ-GRK2-Mediated FAK Phosphorylation at Tyrosine 925 to Regulate ERK Activation Involving Neuronal Focal Adhesions.

doi: 10.3389/fncel.2020.00176

Figure Lengend Snippet: FIGURE 5 | CB1-stimulated FAK phosphorylation at tyrosine 925 and ERK2 phosphorylation at tyrosine 204 are mediated by Gβγ and GRK2 in N18TG2 cells. (A–D) Cells were pretreated with the Gi/o inhibitor pertussis toxin (100 ng/mL, 20 h), Gβγ inhibitor gallein (10 µM, 15 min), or GRK2 inhibitor (1 µM, 15 min) before treatment with 0.01 µM WIN55212-2 (WIN) for 1 or 2 min (m) at 37◦C. Cell lysates were analyzed using western blots and representative blot images are shown. Data are reported as mean ± SEM of (B) % of vehicle-treated FAK 925 Tyr-P (normalized to total FAK levels) at the same time point, (C) % of vehicle-treated ERK2 204 Tyr-P (normalized to total ERK2 levels) at the same time point, and (D) % of basal/time 0 ERK2 204 Tyr-P (normalized to total ERK2 levels). Significance was assessed using One Way ANOVA followed by Dunnett’s multiple comparisons posthoc test [*p < 0.01, #p < 0.05 indicates significantly different from vehicle-treated (at the same time point); **p < 0.05 indicates significantly different from basal/time 0]. (E–H) Cells (2 × 105) were transfected with no siRNA (mock transfection), GRK2-specific siRNA (100 nM), or negative control siRNA (100 nM) before treatment with 0.01 µM WIN for 2 min at 37◦C. Immunoblot analysis was performed and data are reported as mean ± SEM of the % change over basal (G) FAK 925 Tyr-P (normalized to total FAK levels) and (H) ERK2 204 Tyr-P (normalized to total ERK2 levels). Significance was assessed using One Way ANOVA followed by Dunnett’s multiple comparisons posthoc test (*p < 0.01, #p < 0.05 indicates significantly different from GRK2 siRNA at the same time point). For each dataset, cells were cultured and experiments were completed on at least three separate occasions.

Article Snippet: N18TG2 cells were transfected with 100 nM GRK2-specific siRNA (mouse) or negative control siRNA-A using siRNA transfection reagent according to the manufacturer’s protocol (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Western Blot, Transfection, Negative Control, Cell Culture

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-content imaging and RNAi screens for investigating kinase network plasticity

doi: 10.1007/978-1-4939-7154-1_10

Figure Lengend Snippet: Pipetting scheme for reverse transfection of adherent tissue culture cells with arrayed siRNA oligonucleotides. Steps either side of the dashed line represent preparation of parallel copies of the library siRNA transfections combined with either non-targeting (NT) siRNA or a specific additional modifier siRNA (siMOD). This siRNA screen can either look for epistatic modifiers of the siMOD target gene or, vice versa, to test a candidate siMOD target for epistatic impact on the various targets in the library. Values in bold and in parentheses indicate the current volume per well of liquid in the plates upon completion of the indicated steps. (A) A plate of an siRNA library arrayed in a 96 well format is diluted in siRNA buffer to yield a 200 nM copy on a new plate containing 70 ul final volume. The siRNA dilution buffer is supplemented with either 55.5 nM NT or siMOD siRNA, or non-supplemented if the screen does not involve a modifier (see Note 1). (B) Lipid-siRNA complexes are generated by adding diluted lipid mix to the plate(s). (C) The resulting complexes are then sub-divided into three copies on new plates, each with opaque walls and a transparent base, to which the cells are subsequently added. Final oligonucleotide concentrations once the cells have been added are 20 nM for the library siRNA and 5 nM for the modifier siRNA.

Article Snippet: HiPerfect siRNA transfection lipid (Qiagen, catalogue number 301704).

Techniques: Transfection, Generated

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: High-content imaging and RNAi screens for investigating kinase network plasticity

doi: 10.1007/978-1-4939-7154-1_10

Figure Lengend Snippet: Example scatter plots of data produced using this analysis strategy. The R-script in Fig. 15 has been used in conjunction with an output Nuclei.csv file from a siRNA screen. The x-axes display GFP-ratio values calculated from the GFP-CDK2 reporter and the y-axes the mean nuclear fluorescence intensity of CyclinA antibody staining in arbitrary units. Positions of the assay thresholds (bold white lines) are set based on observations of a relevant test set of wells present on each plate of the screen and numbers in each of the quadrants represent the percent cells of the plotted population contained therein. (A) Individual cell assay values from a test set of siRNA representing NT oligonucleotide, siRNA targeting Cyclin A and a siRNA mixture targeting the G1 phase cyclin-dependent kinases CDK6 and CDK4. The Cyclin A knockdown data was used to set an arbitrary threshold for Cyclin A expression positivity/negativity (horizontal white line) whereas the rightwards trend of the double CDK4 and CDK6 knockdown data, resulting in enrichment of CDK2 negative G1 cells, was used to estimate a suitable value for the vertical threshold of the GFP-CDK2 reporter. (B) Example screen data where a candidate epistatic modifier (siMOD) is shown cancelling loss of CDK2 activity and Cyclin A in cells, otherwise driven by loss of CDK6. Non-targeting siRNA control and CDK6 siRNA data values in the absence (upper panels) and presence (lower panels) of a candidate epistatic siMOD siRNA are shown, respectively. NT siRNA substitutes for the absent siMOD siRNA in the upper panel data such that the total siRNA concentration is constant across all transfection conditions in the screen.

Article Snippet: HiPerfect siRNA transfection lipid (Qiagen, catalogue number 301704).

Techniques: Produced, Fluorescence, Staining, Expressing, Activity Assay, Concentration Assay, Transfection