Journal: International Journal of Molecular Sciences

Article Title: Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots

doi: 10.3390/ijms23031195

Figure Lengend Snippet: List of eight commercially available antibodies examined in this study, targeting the ubiquitously expressed human GRK isoforms. Overview of the tested antibodies (monoclonal (mc), polyclonal (pc)) and the tested GRK isoform is provided, including the supplier’s information, our review of each antibody, and the dilution used in Western blot to determine the antibody specificity. Additionally, the number of amino acids (aa) and the calculated approximate molecular weight (MW) for each human GRK isoform are listed. Accession numbers for protein isoforms can be found in the Materials and Methods section and additionally summarized in Supplementary Table S1 .

Article Snippet: GRK6-1 , 576 aa 66 kDa , , , , , , , PB9709 rabbit pc , Boster Biological Technology , C-terminus of human GRK6 (amino acids 382-417) , human, rat , 1:1000 , strong background around 55kDa; can detect human isoform 6-4.

Techniques: Western Blot, Molecular Weight

Journal: Molecular pain

Article Title: Overexpression of GRK6 attenuates neuropathic pain via suppression of CXCR2 in rat dorsal root ganglion.

doi: 10.1177/1744806916646381

Figure Lengend Snippet: Figure 1. Enhanced neuropathic pain and decreased expression of GRK6 in CCI rats. (a) CCI induces a significant decrease in PWT from 3 days to 21 days after surgery in the ipsilateral side. *p <.05, compared with Sham group. (b) The development of heat hyperalgesia from 3 days to 21 days was induced by CCI in the ipsilateral side. *p <.05, compared with Sham group. (c) Expression of GRK6 protein in DRGs was decreased from 3 days to 21 days after surgery in the ipsilateral side with the lowest level seven days after CCI. *p <.05, compared with Sham group. (d) GRK6 mainly expressed in neurons co-localized with b-tubulin, IB4, and CGRP in DRGs, but not in glial cells stained with GFAP. Bar ¼ 75 mm.

Article Snippet: The membrane was probed with antibodies against GRK6 and CXCR2 (antirabbit, 1:100, Boster, P.R.

Techniques: Expressing, Staining

Journal: Molecular pain

Article Title: Overexpression of GRK6 attenuates neuropathic pain via suppression of CXCR2 in rat dorsal root ganglion.

doi: 10.1177/1744806916646381

Figure Lengend Snippet: Figure 2. Suppression of neuropathic pain by GRK6 overexpression. (a) GFP image showed the successfully infection of lentiviral in DRG by sciatic nerve injection. (b) Western blot analysis presented the enhanced expression of GRK6 in DRG from CCI rats after GRK6 lentiviral injection. (c) GRK6 lentiviral injection, rather than NC lentiviral, markedly increased PWT on the CCI rats. *p <.05, compared with CCI group. (d) GRK6 lentiviral injection, rather than NC lentiviral, markedly increased PWL on the CCI rats. *p <.05, compared with CCI group. LV-GFP: lentiviral carrying the green fluorescent protein; CCI: chronic constriction injury; NC-LV: Negative control lentivirus; PWL: paw withdraw latency; PWT: paw mechanical withdrawal thresholds.

Article Snippet: The membrane was probed with antibodies against GRK6 and CXCR2 (antirabbit, 1:100, Boster, P.R.

Techniques: Over Expression, Infection, Injection, Western Blot, Expressing, Negative Control

Journal: Molecular pain

Article Title: Overexpression of GRK6 attenuates neuropathic pain via suppression of CXCR2 in rat dorsal root ganglion.

doi: 10.1177/1744806916646381

Figure Lengend Snippet: Figure 4. Reversal of hyperexcitability of DRG neurons in CCI rats by GRK6 overexpression. (A) Bright-field (a), GRK6 LV-GFP (b), DiI fluorescence (c), and merge of GFP and Dil fluorescence (d) images of acutely dissociated DRG neurons. A hind paw innervating DRG neuron infected by GRK-LV is indicated by arrow. Bar ¼ 50 mm. (B) GRK6 overexpression significantly hyperpolarized the RP of hind paw innervating DRG neurons. *p <.05 vs. NC-LV. (C) GRK6 overexpression notably depolarized the AP threshold (*p <.05 vs. NC-LV). (D) GRK6 overexpression also markedly increased rheobase. *p <.05 vs. NC-LV. (E, G) Examples of APs by 100 and 300 pA ramp current injection from NC-LV and GRK6-LV-infected DRG neurons. (F, H) Bar graph showed a significant decrease in numbers of APs evoked by 100 and 300 pA ramp current stimulation by GRK6 overexpression. *p <.05 vs. NC-LV. RP: resting membrane potential; AP: action potential; NC-LV: negative control lentivirus.

Article Snippet: The membrane was probed with antibodies against GRK6 and CXCR2 (antirabbit, 1:100, Boster, P.R.

Techniques: Over Expression, Fluorescence, Infection, Injection, Membrane, Negative Control

Journal: Molecular pain

Article Title: Overexpression of GRK6 attenuates neuropathic pain via suppression of CXCR2 in rat dorsal root ganglion.

doi: 10.1177/1744806916646381

Figure Lengend Snippet: Figure 5. Reversal of CXCR2 upregulation by GRK6 overexpression in CCI rats. Western blot assays demonstrated a significant upregulation of CXCR1 (a), CXCR2 (b), and CXCR4 (c) protein expression seven days after CCI. *p <.05 vs. Sham. GRK6 overexpression remarkably downregulated CXCR2 protein expression (e) but not CXCR1 (d) and CXCR4 (f) protein expression in CCI rats. *p <.05 vs. NC-LV. CCI: chronic constriction injury; NC-LV: negative control lentivirus.

Article Snippet: The membrane was probed with antibodies against GRK6 and CXCR2 (antirabbit, 1:100, Boster, P.R.

Techniques: Over Expression, Western Blot, Expressing, Negative Control

Journal: Molecular pain

Article Title: Overexpression of GRK6 attenuates neuropathic pain via suppression of CXCR2 in rat dorsal root ganglion.

doi: 10.1177/1744806916646381

Figure Lengend Snippet: Figure 6. Correlation of GRK6 and CXCR2 expression and attenuation of neuropathic pain by CXCR2 inhibitor. (a) Expression of CXCR2 protein in DRGs was increased from day 3 to day 21 following CCI. *p <.05 vs. Sham. (b) Correlation analysis showed that the alternations of CXCR2 and GRK6 following CCI were negatively correlated. r2¼ 0.824, p ¼.042. (c) Co-immunoprecipitation showed the co-localization of CXCR2 and GRK6 in DRGs in CCI rats. (d) Immunofluorescence analysis showed CXCR2 was co-expressed in GRK6 positive DRG neurons. GRK6-positive cells (top left) and b-tubulin-positive cells (bottom left) shown in green. CXCR2-positive cells were shown in red (middle column). Merge of double labeling of GRK6 and CXCR2 (top right) and merge of b-tubulin positive staining and CXCR2 labeling (bottom right). Scale bar ¼ 50 mm. (e, f) Administration of the selective CXCR2 antagonist SB225002 mitigated mechanical hyperalgesia and heat hyperalgesia in CCI rats. Antinociceptive effects of a single intrathecal (it.) injection of 20 mg SB225002 were observed at 1 h and disappeared at 2 h. Antinociceptive effects induced by a single injection of SB225002 at 40 mg were observed at 1 h and disappeared 6 h after injection. Injection of SB225002 (10 mg, i.t.) did not alter the PWT and PWL of CCI rats. N ¼ 6 rats for each group, *p <.05, compared to NS group. PWL: paw withdraw latency; PWT: paw mechanical withdrawal thresholds.

Article Snippet: The membrane was probed with antibodies against GRK6 and CXCR2 (antirabbit, 1:100, Boster, P.R.

Techniques: Expressing, Immunoprecipitation, Immunofluorescence, Labeling, Staining, Injection