Fig 2: GRK isoform specificity of several commercially available GRK antibodies. HEK293 cells were transiently transfected with pcDNA3 plasmids encoding either GRK2, GRK3-1, GRK3-2, GRK5, GRK6-1, GRK6-2, GRK6-3, or GRK6-4 as indicated. GRK isoform specificity of indicated GRK antibodies was investigated in Western blot analysis. Representative blots are shown. Two different antibodies raised against GRK2 (a,b), GRK3 (c,d), GRK5 (e,f), and GRK6 (g,h) were used for the analysis. Approximate molecular weight of each antigen is stated. Detailed information on each antibody examined is listed in Table 1. Actin served as loading control.

Fig 3: GRK6 localization in MIN6 cells. nt-shRNA cells were infected with the indicated lentiviruses at 2 MOI. After 24 h, the cells were trypsinized and moved to new plates with sterile coverslips for an additional 48 h. The cells were then fixed, permeabilized, blocked, and stained with rabbit anti-GRK6 primary antibody and Alexa-Fluor 488–conjugated anti-rabbit secondary antibody. Images were taken using a Nikon A1R fluorescent microscope in the Sidney Kimmel Cancer Center bioimaging facility. NC is the negative control virus.

Fig 4: Effects of transcriptional or translational inhibition on GRK6 mRNA expression.(A) Treatment with actinomycin D causing an inhibition of transcriptional activity in serum starved Jurkat cells over 6 hours led to an increase in mRNA expression in comparison to treatment with 100 nM PMA only. Endogenous GRK6 mRNA expression was detected using the comparative 2-ΔΔCt method and vehicle control with DMSO. N = 3, measured in duplicates, mean + standard error of the mean. (B) Depiction of the relative mRNA expression, shown as 2-Δ Ct values in comparison to the reference genes HPRT and B2M. Treatment with actinomycin D resulted in a general decrease in GRK6 mRNA expression. Statistics: Ordinary one-way ANOVA + Sidak’s multiple comparisons test, ***p<0,001. Depicted: mean + standard error of the mean. (C) Inhibition of translational activity by means of cycloheximide over 6h in serum starved Jurkat cells caused a decrease in mRNA expression. GRK6 mRNA expression was calculated using the comparative 2-ΔΔCt method and vehicle control with DMSO. Mann-Whitey-U-test, **p = 0,0043. N = 3, measured in duplicates. Depicted: mean + standard error of the mean. (D) Depiction of the relative mRNA expression, shown as 2-Δ Ct values in comparison to the reference genes HPRT and B2M. Statistics: Ordinary one-way ANOVA + Sidak’s multiple comparisons test. *p = 0,01, **p = 0,008, ***p = 0,0002. Depicted: mean + standard error of the mean.

Fig 5: GRK6 rescue in knockdown cells restores insulin secretion after high glucose treatment.A, GRK6 knockdown cells were infected with lentivirus encoding human GRK6 at 2 MOI for 24 h. Each infected well was resuspended and transferred to three new wells and allowed to grow for an additional 48 h. The cells were put in low glucose (0.01 mM) KRBH buffer to equilibrate insulin levels for 30 min, washed, and KRBH buffer containing 0.01 to 10 mM glucose was added for 2 h. The level of GRK6 expression was determined by lysing the cells and Western blotting using an anti-GRK6 antibody. B, insulin secretion was measured by ELISA as described in Experimental procedures prior to lysis. Statistical significance was determined using two-way ANOVA with a post hoc Tukey’s multiple comparisons test. The data are represented by the mean ± SD from three to four independent experiments. C, GRK6 knockdown cells were infected with 1.7 MOI lentivirus encoding human GRK6 for 48 h. Each infected well was resuspended and transferred to two new wells and allowed to grow for an additional 24 h. The level of GRK6 expression was evaluated by Western blotting. D, cells were equilibrated in low glucose (0.01 mM) KRBH buffer for 30 min and then washed and treated with either low glucose buffer (−) or complete medium (+) for 1 h. Cell media was removed and assayed by ELISA to determine the level of secreted insulin. Statistical significance was determined by two-way ANOVA. The data are represented by the mean ± SD from three independent experiments. Ponceau staining was used as a protein loading control. KRBH, Krebs Ringer bicarbonate buffer with Hepes; MOI, multiplicity of infection.