Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: Aggregated phospho-S670-GRK2 on dysfunctional mitochondria of aged AD mice (A) Immunoblot of GRK2 in hippocampal lysates of 18-month-old Tg2576 (AD) and B6 mice. GNAO served as loading control. The lower panel shows quantitative data. (B and C) Immunofluorescence of GRK2 (green) and mitochondrial TOMM40 (red) in the CA1 hippocampal area of 18-month-old Tg2576 (left) and B6 (right) mice. Upper panels show the area marked with a white square frame in a 5-fold higher magnification (scale bars: 20 μm). Nuclei are stained with DAPI (blue). Data are from n = 4 mice per group. Replicates are shown in A and S1B. Quantitative data are shown in (C) and E–S1H. (D–G) Immunoblot of hippocampal cytosolic (D and F) and mitochondrial (E and G) GRK2 and phospho-S670-GRK2 (p-S670) in 18-month-old Tg2576 (AD-18mo), 18-month-old B6 (B6-18mo), and 12-month-old Tg2576 (AD-12mo) mice. Cytosolic PPP2CA was determined (D and F). ADSL and TOMM40 are loading controls. (H) Immunoblot of hippocampal mitochondrial Aβ (IB: Aβ) in 18-month-old Tg2576 (AD-18mo), B6 (B6-18mo), and 12-month-old Tg2576 (AD-12mo) mice. Arrowheads mark monomeric Aβ peptides and APP. TOMM40 is the loading control. (I and J) Hippocampal mitochondrial ACO2 and reactivated ACO2 activity (I) and mitochondrial ATP (J) of 12- and 18-month-old Tg2576 and B6 mice. Data are mean ± SD; n = 4 mice per group (A, C, D, F, I, and J) or n = 4 biological replicates (E, G, and H; hippocampi from three mice were pooled for one isolation, which is defined as a biological replicate); unpaired, two-tailed t test [df = 6; t = 9.179 (A); 3.613, 1.357, 0.3557, 5.674 (C); 3.833, 22.22, 10.66 (D); 6.815, 2.083, 9.466, 2.817 (E); 2.806, 11.00, 4.495 (F); 11.47, 18.28, 11.65, 23.38 (G)]; one-way ANOVA and Tukey’s test [F(2,9) = 55.77 (H); F(3,12) = 113.9, 12.37 (I); 21.54 (J)]. See also A, S1B, S1E–S1H, and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Activity Assay, Isolation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: TOMM6 is an interaction partner of phospho-S670-GRK2 (A) Immunoblot of GRK2 in brain lysates of 18- and 12-month-old Tg2576 AD mice. Data are mean ± SD; n = 4 mice; (unpaired, two-tailed t test; df = 6, t = 20.02). (B and C) Immunoaffinity enrichment of phospho-S670-GRK2 (AP: p-S670, lanes 1–4) from 18-month-old (B; lanes 1 and 2) and 12-month-old (C; lanes 3 and 4) Tg2576 mouse brain mitochondria followed by immunoblot detection (IB) of enriched phospho-S670-GRK2 (left) and co-enriched TOMM6 (right). Lane 5 (C) shows a control immuno-affinity matrix (con). Two biological replicates are from 18-month-old mice, and three biological replicates are from 12-month-old Tg2576 mice. (D) Immunoaffinity enrichment of phospho-S670-GRK2 (AP: p-S670-GRK2) from brain mitochondria of 12-month-old Tg2576 mice and nano-LC-ESI-MS/MS identification of co-enriched TOMM6. (E) Immunoblot (IB) of TOMM6 (upper) and TOMM40 (lower) on brain mitochondria from 18-month-old Tg2576 and B6 mice. The right panel shows quantitative data. (F) Immunoaffinity enrichment of TOMM6 (AP: TOMM6) from brain mitochondria of 18-month-old Tg2576 (AD) and B6 (B6) mice, followed by immunoblot detection (IB) of enriched TOMM6 (left) and co-enriched TOMM40 (middle). The right panel shows quantitative data. (E and F) Data are mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test [df = 6; t = 0.06099 (E), 12.59 (F)]. (G and H) In vitro phosphorylation of TOMM6 by GRK2 or p-S670-GRK2 (pS670). TOMM6-ST-AA was not phosphorylated by GRK2. (H) shows quantitative data; mean ± SD; n = 4 biological replicates; one-way ANOVA and Tukey’s test; F(2,9) = 83.12, 206.5. (I) MDS data of TOMM6 showing the frequency distribution of the unfolded molecular weight (MW) of TOMM6 ( n = 40 measurements; left) and the linear regression analysis of the relationship between unfolded MW and TOMM6 (right). (J) Binding energies (E_RDOCK) of monomer-monomer docking, yielding dimers, and tetramer-tetramer docking yielding octamers, of wild-type TOMM6 (WT) and phosphomimetic TOMM6-ST-DD. Data represent means with range; one-way ANOVA and Tukey’s test [F(3,20) = 7.127]. (K) Immunoblot (IB) of TOMM6 in primary neurons expressing wild-type TOMM6 (WT) or TOMM6-ST-DD. Neurons were treated without and with aggregated Aβ and the GRK2 inhibitor, CMPD101 [+Aβ (immunoblot), +Aβ+101 (bar graph)]. Data represent mean ± SD; n = 4 biological replicates; one-way ANOVA and Tukey’s test [F(3,12) = 2141]. See also .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Western Blot, Two Tailed Test, Control, Tandem Mass Spectroscopy, In Vitro, Phospho-proteomics, Molecular Weight, Binding Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: Inactive GRK2 enhances TOMM6 aggregation, Aβ plaques, and mortality, whereas wild-type GRK2 is beneficial (A) Immunoblot of hippocampal cytosolic GRK2 in 16-month-old Tg2576- GRK2S670D (S670D) and Tg2576 (AD16mo) mice (upper blot). The control blot detects ADSL (second blot). The third blot shows GRK2 in hippocampal lysates from 16-month-old Tg2576- GRK2S670D (S670D) and 16-month-old Tg2576- GRK2 (GRK2) mice. The lower control blot detects GNAO. (B) Immunoblot of hippocampal cytosolic GRK2 (upper) and p-S670-GRK2 (middle) in 16-month-old Tg2576- GRK2K220R (K220R), Tg2576- GRK2 (GRK2), and Tg2576- GRKInh (Inh) mice. ADSL is the cytosolic loading control. (A and B) Data represent mean ± SD; n = 4 mice per group; unpaired, two-tailed t test (A; df = 6, t = 5.640, 9.369, 4.242); one-way ANOVA and Tukey’s test [B; F(2,9) = 7.991, 31.84]. (C) Immunoblot of hippocampal mitochondrial GRK2 (upper) and mitochondrial TOMM6 (lower) in 16-month-old Tg2576- GRK2S670D (S670D) and Tg2576 (16 mo) mice. TOMM40 is a loading control (middle panel). (D–F) Immunoblot of hippocampal mitochondrial GRK2 (D and E) and TOMM6 (F) in 16-month-old Tg2576- GRK2K220R (K220R), Tg2576- GRK2 (GRK2), and Tg2576- GRKInh (Inh) mice. TOMM40 is the loading control (E, lower panel). (D and F) present quantitative data. (C, D, and F) Data are mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test (C; df = 6, t = 7.403, t = 10.67, t = 4.247); one-way ANOVA and Tukey’s test [D,F; F(2,9) = 38.19, 137.0]. (G) Hippocampal mitochondrial ACO2 activity without (left) and with reactivation (right) in 16-month-old Tg2576- GRK2S670D (S670D), Tg2576- GRK2K220R (K220R), Tg2576- GRKInh (GRKInh), and Tg2576- GRK2 (GRK2) mice in comparison to 16-month-old Tg2576 (AD16mo) mice. Data are mean ± SD; n = 4 mice per group; one-way ANOVA and Tukey’s test; F(4,15) = 63.00 (left), 4.111 (right). (H) Probability of survival of the indicated transgenic mouse lines with Kaplan-Meier survival analysis and log rank (Mantel-Cox) test; n = 103 male and female mice ( n = 136 for Tg- GRK2K220R ); df = 1; chi-square 4.940 and p = 0.0264 for Tg2576- GRK2 vs. Tg2576; chi-square 16.83 and p < 0.0001 for Tg2576- GRK2S670D vs. Tg2576; df = 1; chi-square 20.70 and p < 0.0001 for Tg- GRK2K220R vs. Tg2576; chi-square 7.459 and p = 0.0063 for Tg2576- GRKInh vs. Tg2576; chi-square 28.51 and p < 0.0001 for Tg2576- GRK2K220R vs. Tg2576. (I–K) Hippocampal γ-secretase (I), β-secretase (J), and α-secretase (K) of 16-month-old Tg2576- GRK2S670D (S670D), Tg2576- GRK2K220R (K220R), Tg2576- GRKInh (GRKInh), and Tg2576- GRK2 (GRK2) mice compared to Tg2576 (AD16mo) mice. Data represent mean ± SD; n = 6 mice per group; one-way ANOVA and Tukey’s test; F(4,25) = 55.73 (I), 44.06 (J), 41.47 (K). (L) Aβ plaque load of histological sections shown in (M); E and C. Data are mean ± SD; n = 6 mice per group; one-way ANOVA and Tukey’s test [left panel; F(3,20) = 12.13]; unpaired, two-tailed t test (right panel; df = 10, t = 4.537). (M) Hippocampal Aβ plaques (brown) of 16-month-old Tg2576- GRK2S670D , Tg2576- GRK2K220R , Tg2576- GRKIn h, and Tg2576 (Tg2576 16mo) mice and 18-month-old Tg2576- GRK2 and Tg2576 (Tg2576 18mo) female (f) and male (m) mice. Counterstaining was performed with hematoxylin (HE). Immunohistology is representative of six mice per group; scale bars: 200 μm. Replicates are shown in E and C. See also and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Western Blot, Control, Two Tailed Test, Activity Assay, Comparison, Transgenic Assay

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: Neuron-specific TOMM6 counteracts insoluble Aβ aggregation but increases soluble Aβ and mortality (A) Plasmid for generation of Tg2576- TOMM6 mice (upper) and PCR genotyping (middle and lower panels); c, control without genomic DNA; P, positive plasmid control; M, DNA marker. Double-transgenic mice are marked with an asterisk. (B–D) Immunoblot of hippocampal mitochondrial TOMM6 (B), GRK2 (C), and phospho-S670-GRK2 (D) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. The control immunoblot (C, lower panel) detects TOMM40. (E and F) Immunoblot of hippocampal, cytosolic GRK2 (E, left) and phospho-S670-GRK2 (E, right) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. The control blot detects ADSL. Quantitative data are shown in (F). (B, C, D, and F) Data are mean ± SD; n = 4 biological replicates per group (B, C, and D) and n = 4 mice per group (F); unpaired, two-tailed t test; df = 6; t = 4.741, 74.19 (B); 10.33, 13.47 (C); 7.329, 8.060 (D); 1.776, 0.3092 (F). (G) Immunofluorescence of GRK2 (green) and TOMM40 (red) in the hippocampal CA1 area of 18-month-old Tg2576 (left) and Tg2576- TOMM6 (right) mice. Nuclei are stained with DAPI (blue). The upper panels show the area marked by a white frame in a 5-fold higher magnification (scale bars: 20 μm). Immunofluorescence is representative of n = 4 mice per group. Replicates and quantitative data are shown in A, S1C, and S1E–S1H. (H and I) Hippocampal mitochondrial ACO2 activity (H) and mitochondrial ATP (I) of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. Data are mean ± SD ( n = 4 mice per group); unpaired, two-tailed t test; df = 6; t = 10.77, 3.346 (H); t = 4.420 (I). (J) Immunohistology of hippocampal Aβ plaques (brown) of 18-month-old Tg2576 and Tg2576- TOMM6 male (m) mice. Nuclei were stained with hematoxylin (HE). Immunohistology is representative of six mice per group (scale bar: 200 μm). Replicates and quantitative data are shown in A and S5B. (K and L) Insoluble (K) and soluble (L) Aβ1-40 and Aβ1-42 in brains of 18-month-old Tg2576 and Tg2576- TOMM6 (+TOMM6) mice. Data are mean ± SD; n = 6 mice per group; unpaired, two-tailed t test; df = 10; t = 12.59, 13.23 (K); 12.06, 9.365 (L). (M) Probability of survival of male and female Tg2576- TOMM6 ( n = 62), Tg2576 ( n = 103), and Tg- TOMM6 mice ( n = 103) was performed by Kaplan-Meier survival analysis with log rank (Mantel-Cox) test; df = 1, chi-square 12.52 and p = 0.0004 for Tg- TOMM6 vs. Tg2576; chi-square 11.7 and p = 0.0006 for Tg2576- TOMM vs. Tg2576. (N) Hippocampal α-, β-, and γ-secretase in 18-month-old Tg2576 and Tg2576- TOMM6 mice. Data represent mean ± SD; n = 6 mice per group (unpaired, two-tailed t test; df = 10, t = 1.519, 2.078, 1.791). See also A, S1C, S1E–S1H, and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Plasmid Preparation, Control, Marker, Transgenic Assay, Western Blot, Two Tailed Test, Immunofluorescence, Staining, Activity Assay

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: Development of a GRK2 function modulator that prevents phospho-S670-GRK2 and TOMM6 aggregation (A) CPD10 ligand affinity chromatography (AP:C10) with brain lysates of 12-month-old Tg2576- GRK2 mice. CPD10-interacting GRK2 (lanes 3 and 4; left) was eluted from the CPD10-containing affinity matrix and detected by immunoblot (IB). TOMM6 did not interact and was below detection limit (lanes 3 and 4; right). Lanes 1 and 2 show a control affinity matrix (AP: Con). (B) Enrichment of p-S670-GRK2(K220R) (lanes 7 and 8; left) and co-enrichment of TOMM6 (lanes 7 and 8; right) by CPD10 ligand affinity chromatography (AP:C10) from brains from 12-month-old Tg2576- GRK2K220R mice. Lanes 5 and 6 show a control affinity matrix. (A and B) Two biological replicates are shown for each transgenic mouse line. (C and D) Immunoaffinity enrichment (AP) of TOMM6 from brain mitochondria of 18-month-old Tg2576 mice without or with CPD10 treatment (±C10) for 6 months followed by immunoblot detection of enriched TOMM6 (IB: TOMM6) and phosphorylated TOMM6 (IB: p-Ser). Representative immunoblots (C) and quantitative data (D) are shown. Data represent mean ± SD; n = 4 biological replicates per group; unpaired, two-tailed t test; df = 6; t = 3.611 (TOMM6 aggr.), 11.74 (TOMM6 monomer), 2.811 (phospho-TOMM6 aggr.), 9.016 (phospho-TOMM6 monomer). (E) Molecular docking of CPD10 (CPK model, red) to GRK2 (upper) and to p-S670-GRK2 (middle) by CDOCKER. Left images show GRK2 and p-S670-GRK2 with modeled C-terminus (red), the large lobe (yellow), the small lobe (blue), and N-terminal and C-terminal domains of GRK2 (gray). The S670 phosphorylation is shown in green (CPK model). Right panels show the docking site of CPD10 in the large lobe of GRK2 (upper) and in the ATP-binding site of p-S670-GRK2 (middle). (F) Binding energies (kcal/mol) of CPD10 (S-isomer) docking to GRK2 and phospho-S670-GRK2. Data are mean of top five poses ± SD: −389.1 ± 12.1; +40.3 ± 32.7; −81.2 ± 30.3. (G) IC 50 values of CPD10 for inhibition of TOMM6 phosphorylation by p-S670-GRK2 and GRK2-S670A (S670A). Data are mean ± SD; n = 4 independent experiments with four technical replicates each. (H) CPD10 (10 μM) did not substantially inhibit GRK2-related kinases. (I) Conceptual model and molecular dynamics simulation of GRK2 function modulation by CPD10. CPD10 could stabilize the GRK2 monomer (cyan, right) and prevent phospho-S670-GRK2 and TOMM6 aggregation (left); OMM: outer mitochondrial membrane; IMS: intermembrane space. Potential energies (E) of GRK2 (lower right) and phospho-S670-GRK2 (p-S670; lower left) without (w/o) and with CPD10 (+C10) interaction are given after energy minimization without (w/o MD sim) and with MD simulation (MD sim). See also and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Affinity Chromatography, Western Blot, Control, Transgenic Assay, Two Tailed Test, Phospho-proteomics, Binding Assay, Inhibition, Membrane

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: GRK2 function modulation counteracts dysfunctional phospho-S670-GRK2 aggregation and retards AD progression and mortality in mice (A and B) Immunoblot of hippocampal, cytosolic GRK2 (A, left) and p-S670-GRK2 (A, right) of 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10; 8 mg/kg/d), and 12-month-old, untreated (12 mo) Tg2576 mice. The control blot detects ADSL. Data are mean ± SD; n = 4 mice; one-way ANOVA and Tukey’s test; F(2,9) = 23.54, 37.45 (B). Below the right panel (A), brain and serum concentrations, and brain-to-serum ratios of CPD10 (mean ± SD; n = 6 mice) are shown. See also G and S5H. (C and D) Immunoblot of hippocampal, mitochondrial GRK2 (C) and TOMM6 (D) of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. The control blot detects TOMM40. Data represent mean ± SD; n = 4 biological replicates; unpaired, two-tailed t test; df = 6; t = 6.179, 1.729 (C); t = 7.540, 20.48 (D). (E and F) Hippocampal mitochondrial ACO2 activity without and with reactivation (E), and ATP contents (F) were determined in 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10), and 12-month-old, untreated (12 mo) Tg2576 mice. Data represent mean ± SD ( n = 4 mice per group); one-way ANOVA and Tukey’s test; F(2,9) = 135.2, 13.11 (E); 30.06 (F). (G and H) Immunoblot of synaptosomal, hippocampal SNAP25 in 18-month-old, untreated (18 mo), CPD10-treated (18mo + CPD10), and 12-month-old, untreated (12 mo) Tg2576 mice. The control blot detects GNAO. Data are mean ± SD ( n = 4 mice per group); one-way ANOVA and Dunnett’s test; F(2,9) = 11.64 (G). (I) Immunofluorescence of mitochondrial TOMM40 (red) and GRK2 (green) in the hippocampal CA1 area of brain specimens from 18-month-old, CPD10-treated (Tg2576 18mo + CPD10) and untreated (Tg2576 18mo) Tg2576 mice. Nuclei were stained with DAPI (blue); scale bars: 20 μm. Immunofluorescence is representative of n = 7 mice per group. Three replicates are shown in . Four replicates are shown in A and S1D. (J) Overrepresentation analysis of significantly up-regulated transcripts (≥2-fold; p < 0.05) in frontal cortices of 18-month-old CPD10-treated Tg2576 mice compared to untreated mice. Biological processes (GO:BP) with p values <0.001 in the overrepresentation analysis and involvement in neurogenesis are shown. p values are presented as negative log10 of adjusted p values. Data are shown in . (K) Heatmap of significantly different ( p < 0.05) transcripts ( NeuN , Gfap , and Cx3cr1 ) in frontal cortices of 18-month-old, CPD10-treated Tg2576 (AD + C10) compared to untreated Tg2576 mice (AD). Data are shown in . (L) Activities of hippocampal α-, β-, and γ-secretase in 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. Data represent mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10, t = 4.303, 5.280, 5.049. (M) Immunohistological detection of Aβ plaques (brown) on coronal brain sections of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) female (f) and male (m) Tg2576 mice. Counterstaining was performed with hematoxylin (HE). Immunohistology is representative of six mice per group (scale bar: 200 μm). Replicates and quantitative data are shown in A and S5B. (N and O) Insoluble (N) and soluble (O) Aβ1-40 and Aβ1-42 in brains of 18-month-old, untreated (18 mo) and CPD10-treated (18mo + CPD10) Tg2576 mice. Data represent mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10; t = 14.91, 16.01 (N); 5.352, 2.937 (O). (P) Probability of survival of CPD10-treated (+CPD10) and untreated Tg2576 mice ( n = 18 mice) was determined by Kaplan-Meier survival analysis with log rank (Mantel-Cox) test; df = 1, chi-square = 6.800. See also A, S1D–S1H, , , and ; and .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Western Blot, Control, Two Tailed Test, Activity Assay, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Analysis of GRK2 aggregation in the pathology of Alzheimer disease in animal models

doi: 10.1016/j.xcrm.2026.102707

Figure Lengend Snippet: A proteasome activity enhancer augments phospho-S670-GRK2 degradation and retards neurodegenerative AD symptoms (A) Binding energy (left), docking site (middle), and molecular docking of CPD57 to PSMA1 (chain E) and PSMA3 (chain F) of the 20S proteasome (8QYM) by CDOCKER. Data (left) are mean ± SD, of top 10 poses. (B) CPD57 ligand affinity chromatography (AP:C57) with brain protein lysates of 6-month-old B6 mice. CPD57-interacting PSMA3 (lanes 1 and 2) was detected in immunoblot (IB). Lanes 3 and 4 are controls from a control affinity matrix (AP: Con.). Two biological replicates are shown (1,3; 2,4). (C) Nano-LC-ESI-MS/MS identification of PSMA3 as a CPD57-interacting protein in brain lysates of B6 mice. (D) Brain contents of CPD57 (left) and brain-to-serum ratios (right) of 18-month-old Tg2576 mice after 6 months of treatment with the indicated doses of C57. Data are mean ± SD ( n = 6 mice per group); one-way ANOVA and Tukey’s test; F(3,20) = 16.93 (left); F(2,15) = 2.695 (right). (E) Enhanced proteasome activity in hippocampi of 18-month-old Tg2576 (AD) mice after 14 days of treatment with CPD57 (AD+57; 10 mg/kg/d). As indicated, the 20S proteasome was activated by SDS (0.03%, +). Proteasome activity is presented as % of the untreated AD control (mean ± SD; n = 4 mice per group); one-way ANOVA and Tukey’s test; F(3,12) = 38.09. (F) Immunoblot (IB) of PSMA3 in hippocampal, cytosolic proteins of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The right panel shows quantitative immunoblot data (df = 6; t = 12.14). (G and H) Immunoblot (IB) of phospho-S670-GRK2 (G, left) and GRK2 with GRK2-specific antibodies against the N-terminus of GRK2 (G, right) in hippocampal cytosolic proteins of 18-month-old Tg2576 AD mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The lower control blot detects ADSL. Bar graphs (H) show quantitative data (df = 6; t = 14.45, 5.535). (I and J) Immunoblot (IB) of phospho-S670-GRK2 (I, left) and GRK2 with GRK2-specific antibodies against the N-terminus (I, right) on hippocampal mitochondria of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). The control blot detects TOMM40. Bar graphs (J) show quantitative data (df = 6; t = 2.805, 2.858; 5.266, 12.40). (K) Immunoblot (IB) of TOMM6 on hippocampal mitochondria of 18-month-old Tg2576 mice without treatment (AD) and with 14 days of treatment with CPD57 (+57). Bar graphs (right panels) show quantitative immunoblot data of aggregated (aggr.) and monomeric (mono.) TOMM6 (df = 6; t = 6.417, 9.231). (F, H, J, and K) Data represent mean ± SD; n = 4 mice per group (F and H) and n = 4 biological replicates per group (J and K); unpaired, two-tailed t test. (L) Immunohistological detection of hippocampal Aβ plaques (brown) on coronal brain sections of 18-month-old Tg2576 male (m) mice without treatment (AD) and with 6 months of CPD57 treatment (+57; 10 mg/kg/d). Nuclei were stained with hematoxylin (HE); scale bar: 200 μm. Immunohistology is representative of six mice per group. Replicates and quantitative data are shown in A and S5B. (M and N) Insoluble (M) and soluble (N) Aβ1-40 (upper) and Aβ1-42 (lower) in brains of 18-month-old Tg2576 mice without (AD) and with 6 months of CPD57 treatment (+57). Data are mean ± SD ( n = 6 mice per group); unpaired, two-tailed t test; df = 10; t = 12.73, 13.34 (M); 4.822, 3.117 (N). (O) Proteasome activities of brains from 18-month-old Tg2576 (AD) mice after 6 months of treatment with the indicated doses of CPD57. (P) Contents of insoluble Aβ1-40 in brains of 18-month-old Tg2576 (AD) mice after 6 months of treatment with the indicated doses of CPD57. (O and P) Data are mean ± SD ( n = 6 mice per group); one-way ANOVA and Tukey’s test; F(3,20) = 34.26 (Suc-LLVY-AMC), 32.49 (Boc-LRR-AMC), 27.87 (Z-LLE-AMC), 40.90 (P). See also .

Article Snippet: Rabbit polyclonal anti-ADRBK1/GRK2 antibodies against the N-terminus of GRK2; Lot WOVUS 31186 , LSBio , LS-C358899.

Techniques: Activity Assay, Binding Assay, Affinity Chromatography, Western Blot, Control, Tandem Mass Spectroscopy, Two Tailed Test, Staining

Journal: Cells

Article Title: Cys340Ser Mutation Abolishing S-Nitrosylation Drives GRK2 Mitochondrial Localization and Dysfunction

doi: 10.3390/cells15050458

Figure Lengend Snippet: Group comparison of mitochondrial GRK2 expression ( A ); comparison of fold increase in GRK2 levels between Ad.GFP- and Ad.GRK2.C340S-infected cells ( B ). Statistical significance was determined by ANOVA followed by Bonferroni’s post hoc test for GRK2/VDAC ( p = 0.0003) and by t -test for GRK2 fold increase ( p = 0.0436). Data are shown as mean ± SEM; * p < 0.05 ( n = 5 per group). Representative immunoblots demonstrating GRK2 protein levels in mitochondrial fractions of AC16 cells infected with Ad.GFP or Ad.GRK2-C340S under normoxic or hypoxia/reoxygenation (H/R) conditions. VDAC served as a mitochondrial loading control. Densitometric analysis was performed using Li-Cor Image Studio software and normalized to the respective loading controls. All samples included in each comparative analysis were run on the same gel. Cropped blots are presented for clarity; uncropped blots with molecular weight markers are provided in .

Article Snippet: The primary antibodies used were as follows: GRK2 (13990-1-AP; ProteinTech, Rosemont, IL, USA), GRK2 (05-465; Sigma Aldrich, St. Louis, MO, USA), Drp1 (8570; Cell Signaling Technology, Danvers, MA, USA), Fis1 (32525; Cell Signaling Technology, Danvers, MA, USA), Mfn1 (14739; Cell Signaling Technology, Danvers, MA, USA), Mfn2 (11925; Cell Signaling Technology, Danvers, MA, USA), Opa1 (80471; Cell Signaling Technology), Pink (6946; Cell Signaling Technology, Danvers, MA, USA), Parkin (4211; Cell Signaling Technology, Danvers, MA, USA), LC3 (3868; Cell Signaling Technology, Danvers, MA, USA), Tom20 (42406; Cell Signaling Technology, Danvers, MA, USA), VDAC1 (sc-390996; Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (sc-32233; Santa Cruz Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Comparison, Expressing, Infection, Western Blot, Control, Software, Molecular Weight

Journal: Cells

Article Title: Cys340Ser Mutation Abolishing S-Nitrosylation Drives GRK2 Mitochondrial Localization and Dysfunction

doi: 10.3390/cells15050458

Figure Lengend Snippet: Group comparison of mitochondrial Drp1 ( A ) and Fis1 ( B ) expressions. Statistical significance was determined by ANOVA followed by Bonferroni’s post hoc test. Data are shown as mean ± SEM; ns, p = 0.2021 for Drp1; *, p < 0.05 ( p = 0.0041) for Fis1 ( n = 5–7 per group). Representative immunoblots demonstrating Drp1 and Fis1 protein levels in mitochondrial fractions and total cell lysates of AC16 cells infected with Ad.GFP or Ad.GRK2-C340S under normoxic or hypoxia/reoxygenation (H/R) conditions. VDAC served as a mitochondrial loading control and GAPDH served as a total protein marker. Densitometric analysis was performed using Li-Cor Image Studio software and normalized to the respective loading controls. All samples included in each comparative analysis were run on the same gel. Cropped blots are presented for clarity; uncropped blots with molecular weight markers are provided in .

Article Snippet: The primary antibodies used were as follows: GRK2 (13990-1-AP; ProteinTech, Rosemont, IL, USA), GRK2 (05-465; Sigma Aldrich, St. Louis, MO, USA), Drp1 (8570; Cell Signaling Technology, Danvers, MA, USA), Fis1 (32525; Cell Signaling Technology, Danvers, MA, USA), Mfn1 (14739; Cell Signaling Technology, Danvers, MA, USA), Mfn2 (11925; Cell Signaling Technology, Danvers, MA, USA), Opa1 (80471; Cell Signaling Technology), Pink (6946; Cell Signaling Technology, Danvers, MA, USA), Parkin (4211; Cell Signaling Technology, Danvers, MA, USA), LC3 (3868; Cell Signaling Technology, Danvers, MA, USA), Tom20 (42406; Cell Signaling Technology, Danvers, MA, USA), VDAC1 (sc-390996; Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (sc-32233; Santa Cruz Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Comparison, Western Blot, Infection, Control, Marker, Software, Molecular Weight

Journal: Cells

Article Title: Cys340Ser Mutation Abolishing S-Nitrosylation Drives GRK2 Mitochondrial Localization and Dysfunction

doi: 10.3390/cells15050458

Figure Lengend Snippet: Group comparison of Mfn1 ( A ), Mfn2 ( B ) and Opa1 ( C ) expressions. Statistical significance was determined by ANOVA followed by Bonferroni’s post hoc test. Data are shown as mean ± SEM; ns, p = 0.4591 for Mfn1; p = 0.1796 for Mfn2; p = 0.0552 for Opa1 ( n = 7–9 per group). Representative immunoblots demonstrating Mfn1, Mfn2 and Opa1 protein levels in total cell lysates of AC16 cells infected with Ad.GFP or Ad.GRK2-C340S under normoxic or hypoxia/reoxygenation (H/R) conditions. GAPDH served as a total protein marker. Densitometric analysis was performed using Li-Cor Image Studio software and normalized to the respective loading controls. All samples included in each comparative analysis were run on the same gel. Cropped blots are presented for clarity; uncropped blots with molecular weight markers are provided in .

Article Snippet: The primary antibodies used were as follows: GRK2 (13990-1-AP; ProteinTech, Rosemont, IL, USA), GRK2 (05-465; Sigma Aldrich, St. Louis, MO, USA), Drp1 (8570; Cell Signaling Technology, Danvers, MA, USA), Fis1 (32525; Cell Signaling Technology, Danvers, MA, USA), Mfn1 (14739; Cell Signaling Technology, Danvers, MA, USA), Mfn2 (11925; Cell Signaling Technology, Danvers, MA, USA), Opa1 (80471; Cell Signaling Technology), Pink (6946; Cell Signaling Technology, Danvers, MA, USA), Parkin (4211; Cell Signaling Technology, Danvers, MA, USA), LC3 (3868; Cell Signaling Technology, Danvers, MA, USA), Tom20 (42406; Cell Signaling Technology, Danvers, MA, USA), VDAC1 (sc-390996; Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (sc-32233; Santa Cruz Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Comparison, Western Blot, Infection, Marker, Software, Molecular Weight

Journal: Cells

Article Title: Cys340Ser Mutation Abolishing S-Nitrosylation Drives GRK2 Mitochondrial Localization and Dysfunction

doi: 10.3390/cells15050458

Figure Lengend Snippet: Group comparison of Parkin ( A ), Pink ( B ), LC3 ( C ) and Tomm20 ( D ) expressions. Statistical significance was determined by ANOVA followed by Bonferroni’s post hoc test. Data are shown as mean ± SEM; * p < 0.05, ** p < 0.01 p = 0.0027 for Parkin; p = 0.0093 for Pink (both bands); p = 0.0043 for LC3; ns, p = 0.6020 for Tomm20 ( n = 8–10 per group). Representative immunoblots demonstrating Parkin, Pink, LC3 and Tomm20 in mitochondrial fractions and total cell lysates of AC16 cells infected with Ad.GFP or Ad.GRK2-C340S under normoxic or hypoxia/reoxygenation (H/R) conditions. VDAC served as a mitochondrial loading control and GAPDH served as a total protein marker. Densitometric analysis was performed using Li-Cor Image Studio software and normalized to the respective loading controls. All samples included in each comparative analysis were run on the same gel. Cropped blots are presented for clarity; uncropped blots with molecular weight markers are provided in .

Article Snippet: The primary antibodies used were as follows: GRK2 (13990-1-AP; ProteinTech, Rosemont, IL, USA), GRK2 (05-465; Sigma Aldrich, St. Louis, MO, USA), Drp1 (8570; Cell Signaling Technology, Danvers, MA, USA), Fis1 (32525; Cell Signaling Technology, Danvers, MA, USA), Mfn1 (14739; Cell Signaling Technology, Danvers, MA, USA), Mfn2 (11925; Cell Signaling Technology, Danvers, MA, USA), Opa1 (80471; Cell Signaling Technology), Pink (6946; Cell Signaling Technology, Danvers, MA, USA), Parkin (4211; Cell Signaling Technology, Danvers, MA, USA), LC3 (3868; Cell Signaling Technology, Danvers, MA, USA), Tom20 (42406; Cell Signaling Technology, Danvers, MA, USA), VDAC1 (sc-390996; Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (sc-32233; Santa Cruz Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Comparison, Western Blot, Infection, Control, Marker, Software, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: GRK phosphorylation drives β-arrestin–independent internalization of chemokine receptor CXCR5

doi: 10.1016/j.jbc.2025.111114

Figure Lengend Snippet: Phosphorylation of CXCR5 is restored by the re-expression of GRK2 and GRK5 in ΔQ-GRK HEK293 cells. A , ΔQ-GRK HEK293 cells transiently expressing either FLAG-CXCR5 (n = 1, 2), HA-CXCR5 (n = 3), and pcDNA, GRK2, or GRK5 were stimulated for 10 min without (−) or with (+) 100 nM CXCL13, followed by anti-HA or anti-FLAG immunoprecipitation and immunoblotting with the indicated antibodies. Representative immunoblots are shown. Two asterisks (∗∗) represents unmodified receptor, while one asterisk (∗) represents a nonspecific band. B and C , densitometric analysis of (A) to quantify receptor phosphorylation. Values for phosphorylation were normalized to receptor levels immunoprecipitated (FLAG-CXCR5 or HA-CXCR5) and then compared with the GRK2 stimulated (+) condition. Data represent the mean ± S.D. from three independent experiments. Data were analyzed by one-way ANOVA followed by Šídák's multiple comparisons test. p values are indicated. GRK, G protein–coupled receptor kinase; CXCR5, C-X-C chemokine receptor 5.

Article Snippet: Antibodies against GRK2 (Cat# 13990-1-AP), GRK5 (Cat# 17032-1-AP), FLAG-tag (DYKDDDDK; Cat# 20543-1-AP), and GAPDH (Cat# 600004-1-Ig) were from Proteintech.

Techniques: Phospho-proteomics, Expressing, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: GRK phosphorylation drives β-arrestin–independent internalization of chemokine receptor CXCR5

doi: 10.1016/j.jbc.2025.111114

Figure Lengend Snippet: CXCR5-stimulated G protein activation in WT or ΔQ-GRK HEK293 cells. A , TRUEPATH model figure; donor tagged (Rluc8) G α , Gβ, Gγ-tagged acceptor tagged (GFP2) subunits are transfected at equal DNA ratios. BRET signal starts high in the inactivated (GDP) bound state; GPCR activation then promotes a decrease in BRET signal as the G α and Gβγ subunits separate when GDP is exchanged for GTP. B and C , WT ( B ) and ΔQ-GRK HEK293 cell lines ( C ) were transiently transfected with FLAG-tagged CXCR5, G i1 -Rluc8, β 3 , and γ 9 -GFP2. Baseline BRET signals were measured for 150 s, followed by the manual addition of varying concentrations of CXCL13 (1–100 nM), and the BRET signal was measured every 30 s for 15 min. The average BRET signal of the baseline was subtracted from the BRET of the CXCL13 response and normalized to receptor expression (ΔBRET). Data are the mean ± S.D. of three independent experiments. D , dose response relationships calculated from the area under the curve (AUC) analysis of data shown in ( B and C ). AUC values were normalized to the HEK293 vehicle condition. Data are the mean ± S.D. of three independent experiments. Where not visible, the error bars fall within the symbol. Data were analyzed by two-way ANOVA followed by Šídák's multiple comparisons test. Asterisks (∗∗) represent p < 0.01. GPCR, G protein–coupled receptor; GRK, G protein–coupled receptor kinase; CXCR5, C-X-C chemokine receptor 5; BRET, bioluminescence resonance energy transfer.

Article Snippet: Antibodies against GRK2 (Cat# 13990-1-AP), GRK5 (Cat# 17032-1-AP), FLAG-tag (DYKDDDDK; Cat# 20543-1-AP), and GAPDH (Cat# 600004-1-Ig) were from Proteintech.

Techniques: Activation Assay, Transfection, Expressing, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: GRK phosphorylation drives β-arrestin–independent internalization of chemokine receptor CXCR5

doi: 10.1016/j.jbc.2025.111114

Figure Lengend Snippet: GRKs are essential for β-arrestin recruitment to CXCR5. A and B , WT and ΔQ-GRK HEK293 cell lines transiently expressing HA- and RLuc8-tagged CXCR5 (HA-CXCR5-Rluc8) and pcDNA (background), β-arrestin1-GFP 10 ( A ) or β-arrestin2-GFP 10 ( B ) were stimulated with varying concentrations of CXCL13. BRET signal is representative of three consecutive reads every 30 s averaged together with background (pcDNA) condition subtracted. AUC values were normalized to the highest concentration of CXCL13 (10 μM) from the parental HEK293 cells. Data represent the mean ± S.D. of three independent experiments. Where not visible, the error bars fall within the symbol. Data were fit using GraphPad prism nonlinear regression model [log[agonist]vs. response (three parameters)]. C , surface expression of FLAG-tagged CXCR5 in ΔQ-GRK HEK293 cells transfected with empty vector (pcDNA), GRK2, or GRK5 normalized to pcDNA condition. D and E , cells were transfected with β-arrestin1-GFP 10 ( D ) or β-arrestin2-GFP 10 ( E ) and BRET signals were measured as in ( A ) and ( B ) and the BRET value for the vehicle treatment of each transfection condition was subtracted from each CXCL13 dose. Data represent the mean ± S.D. of three independent experiments. Where not visible, the error bars fall within the symbol. Data were fit using GraphPad prism nonlinear regression model [log[agonist]vs. response (three parameters)]. F , representative immunoblots with indicated antibodies. G – I , kinetic profile of β-arrestin recruitment to CXCR5 by bystander recruitment of NLuc-tagged β-arrestin1 ( G ) or β-arrestin2 ( H ) to plasma membrane–targeted Venus (Venus-CAAX). Baseline BRET signals were measured for 200 s, followed by the manual addition of vehicle or CXCL13 (100 nM) and the BRET signal was measured every 10 s for 20 min. The average BRET signal of the baseline was subtracted from the BRET response of each condition. Data were further normalized by subtracting the vehicle values at each time point from the CXCL13-stimulated values in each transfection condition (ΔBRET). I , area under the curve (AUC) analysis of data shown in ( G and H ). AUC values were normalized to the FLAG-CXCR5 surface expression in each condition, measured by ELISA in parallel with BRET experiments. Data are the mean ± S.D. of three independent experiments. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. p values are shown; values < 0.05 were considered significant. Asterisks (∗) indicates nonspecific band. GRK, G protein–coupled receptor kinase; CXCR5, C-X-C chemokine receptor 5; BRET, bioluminescence resonance energy transfer.

Article Snippet: Antibodies against GRK2 (Cat# 13990-1-AP), GRK5 (Cat# 17032-1-AP), FLAG-tag (DYKDDDDK; Cat# 20543-1-AP), and GAPDH (Cat# 600004-1-Ig) were from Proteintech.

Techniques: Expressing, Concentration Assay, Transfection, Plasmid Preparation, Western Blot, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: GRK phosphorylation drives β-arrestin–independent internalization of chemokine receptor CXCR5

doi: 10.1016/j.jbc.2025.111114

Figure Lengend Snippet: GRKs are essential for agonist-stimulated internalization of CXCR5. A – E , ΔQ-GRK HEK293 cells were transiently transfected with FLAG-CXCR5-NLuc and either Venus targeted to the plasma membrane (PM-Venus) or early endosomes (EE-Venus) plus either empty vector (pcDNA), WT GRK2 and GRK5, or kinase-dead mutants of GRK2 (K220R) and GRK5 (K215R). Baseline BRET signals were acquired for 5 min, followed by the manual addition of vehicle or CXCL13 (100 nM) and BRET measurements were recorded every 30 s for 25 to 30 min at 37 °C. The average BRET signal of the baseline was subtracted from the BRET response of CXCL13 or vehicle, followed by subtracting the vehicle BRET from the CXCL13 BRET (ΔBRET). ΔBRET was normalized to the maximum response of WT GRK2 or GRK5 (norm-ΔBRET). Data represent the mean ± S.D. of three independent experiments. E and F , area under the curve (AUC) analysis. AUC values were normalized to their respective WT condition. Data represent the mean ± S.D. of three independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. Asterisks represent adjusted p values: ∗, p < 0.05; ∗∗, p < 0.001. G and H , representative immunoblots of GRK2 and GRK5 expression. Asterisk (∗) represents, nonspecific band ( H ). GRK, G protein–coupled receptor kinase; CXCR5, C-X-C chemokine receptor 5; BRET, bioluminescence resonance energy transfer.

Article Snippet: Antibodies against GRK2 (Cat# 13990-1-AP), GRK5 (Cat# 17032-1-AP), FLAG-tag (DYKDDDDK; Cat# 20543-1-AP), and GAPDH (Cat# 600004-1-Ig) were from Proteintech.

Techniques: Transfection, Clinical Proteomics, Membrane, Plasmid Preparation, Comparison, Western Blot, Expressing, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: GRK phosphorylation drives β-arrestin–independent internalization of chemokine receptor CXCR5

doi: 10.1016/j.jbc.2025.111114

Figure Lengend Snippet: Analysis of CXCR5 internalization under acute pharmacological inhibition of GRK2 and GRK5. A – D , HEK293 cells were transiently transfected with FLAG-tagged CXCR5-NLuc and either Venus targeted to the plasma membrane (PM-Venus) or early endosomes (EE-Venus). Cells were pretreated with vehicle (DMSO), 10 μM CMPD101 (GRK2/3 inhibitor), or 10 μM CCG273441 (GRK5/6 inhibitor) kinase inhibitors for 15 min at 37 °C before acquiring baseline BRET signals for 5 min, followed by the manual addition of vehicle or CXCL13 (100 nM) and BRET measurements every 30 s for 25 min at 37 °C. The average BRET signal of the baseline was subtracted from the BRET response of CXCL13 or vehicle, followed by subtracting the vehicle BRET from the CXCL13 BRET (ΔBRET). The faint gray dashed line represents the DMSO control ( A ). Data represent the mean ± S.D. of three independent experiments. E and F , area under the curve (AUC) analysis. AUC values were normalized to their respective DMSO conditions. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. G , surface expression of FLAG-tagged CXCR5-NLuc in each transfection condition was measured by parallel ELISA and compared using an unpaired Student’s t test. p values are indicated and ns represents not significant. GRK, G protein–coupled receptor kinase; CXCR5, C-X-C chemokine receptor 5; BRET, bioluminescence resonance energy transfer.

Article Snippet: Antibodies against GRK2 (Cat# 13990-1-AP), GRK5 (Cat# 17032-1-AP), FLAG-tag (DYKDDDDK; Cat# 20543-1-AP), and GAPDH (Cat# 600004-1-Ig) were from Proteintech.

Techniques: Inhibition, Transfection, Clinical Proteomics, Membrane, Control, Comparison, Expressing, Enzyme-linked Immunosorbent Assay, Bioluminescence Resonance Energy Transfer

Journal: The Journal of Biological Chemistry

Article Title: GRK phosphorylation drives β-arrestin–independent internalization of chemokine receptor CXCR5

doi: 10.1016/j.jbc.2025.111114

Figure Lengend Snippet: Analysis of GRK2 and GRK5 re-expression on the internalization of CXCR5 phospho-site cluster variants in ΔQ-GRK HEK293 cells. A , schematic representation of the carboxyl-terminal tail of CXCR5 with the designated phospho-site clusters. Double cluster variants and the triple cluster variant (CTST) are indicated. The amino acid range of each cluster is indicated. Ser/Thr residues are mutated to Ala within each cluster. B and C , ΔQ-GRK cells transiently expressing FLAG-tagged WT CXCR5 and single cluster variants (Dis; Med, Prox) ( B ) or double cluster (Prox/Med; Prox/Dis; Med/Dis) and triple cluster (CTST) variants ( C ), plus empty vector (pcDNA), GRK2, or GRK5. Cells were stimulated with vehicle or 100 nM CXCL13 for 30 min at 37 °C and cell surface receptor was measured by ELISA, as described in . Internalization was calculated as a percent decrease in the background-adjusted absorbance values from CXCL13-stimulated cells relative to vehicle-treated cells. Data represent the mean ± S.D. from three independent experiments. Data were analyzed with one-way ANOVA with Dunnett’s multiple comparisons comparing the internalization of GRK2 or GRK5 add-back between the pcDNA of its respective CXCR5 variant. p values are indicated, and ns denotes not significant. D and E , representative immunoblots with the indicated antibodies are shown. Asterisk (∗) represents, nonspecific band. GRK, G protein–coupled receptor kinase; CXCR5, C-X-C chemokine receptor 5.

Article Snippet: Antibodies against GRK2 (Cat# 13990-1-AP), GRK5 (Cat# 17032-1-AP), FLAG-tag (DYKDDDDK; Cat# 20543-1-AP), and GAPDH (Cat# 600004-1-Ig) were from Proteintech.

Techniques: Expressing, Variant Assay, Plasmid Preparation, Cell Surface Receptor Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Neuroscience Research

Article Title: G Protein‐Coupled Receptor Kinase 5 ( GRK5 ) Modulates Nociceptin/Orphanin FQ Opioid ( NOP ) Receptor Desensitization in Rat Sympathetic Neurons

doi: 10.1002/jnr.70110

Figure Lengend Snippet: Detection of GRK2 and GRK5 expression levels 96 h post‐transfection with GRK2 or GRK5 or GRK2&5 siRNA. Immunofluorescence imaging reveals SG neurons express GRK2 and GRK5. Shown are fluorescence images of SG neurons fixed, permeabilized, and stained with primary antibodies against GRK2, followed by Alexa Fluor 488‐conjugated IgG secondary antibody. The neurons were imaged at 20× with a filter set containing an excitation filter at 480 ± 15 nm, a dichroic beam splitter of 505 nm (LP), and an emission filter at 535 ± 20 nm. The images were pseudocolored; scale bars represent 100 μm. (A) Relative expression of GRK2 (●) and GRK5 (▲) mRNA in SG tissue 96 h post‐transfection with either GRK2 siRNA or GRK5 siRNA alone or combined (B). Quantitative RT‐PCR was carried out with total RNA from scrambled siRNA‐ and GRK siRNA‐transfected SG tissue. The fold‐differences were computed by double delta C t analysis: First, the differences between the GRK and GAPDH expression levels were calculated for each sample (∆ C t ), then the differences between the experimental and control groups were calculated (∆∆ C t ) and fold‐change determined according to the expression 2 − ∆ ∆ C t . * indicates p = 0.013 for GRK2 siRNA, while p values for GRK5 and GRK2&GRK5 siRNA groups were 0.138 and 0.233, respectively, and not significantly (NS) different employing the paired t ‐test. Each point represents one animal. Representative Western blots for GRK2 (C), GRK5 (D), and GRK2&5 (E) in SG tissue 96 h post‐transfection with scrambled (Scr) or GRK2 or GRK5 or both siRNA. The 69 and 64 kDa bands represent GRK2 and GRK5, respectively. Each lane was loaded with 0.5 μg protein and represents two SG isolated from one animal. Both anti‐GRK2 and anti‐GRK5 antibodies were used at a dilution of 1:10 in the Wes system. The summary dot plot (F) shows the mean (±SD) protein levels for GRK2 and GRK5 in SG neurons transfected with GRK2, GRK5, and GRK2&5 siRNA. GRK protein levels were corrected by normalization of band area values to total protein levels for each sample. Relative expression levels between groups were computed by normalizing corrected experimental band area values to the corresponding average corrected band area of the control group. The p ‐values for GRK2, GRK5, and GRK2&GRK5 siRNA groups were 0.197, 0.307, and 0.079, respectively, and not significantly (NS) different employing the paired t ‐test.

Article Snippet: Following size separation, the presence of GRK2 and GRK5 proteins was detected through chemiluminescence using a mouse monoclonal anti‐GRK2 primary antibody (Santa Cruz Biotechnology, cat. #sc‐13143), mouse monoclonal anti‐GRK5 primary antibody (Santa Cruz Biotechnology, cat. #sc‐518005), and a Horseradish Peroxidase‐conjugated anti‐rabbit secondary antibody provided by the manufacturer (Protein Simple, cat. #DM‐001).

Techniques: Expressing, Transfection, Immunofluorescence, Imaging, Fluorescence, Staining, Quantitative RT-PCR, Control, Western Blot, Isolation