snap surface block  (New England Biolabs)


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    Structured Review

    New England Biolabs snap surface block
    (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared <t>to</t> <t>non-internalized</t> control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing <t>SNAP-CCR2</t> that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap surface block/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap surface block - by Bioz Stars, 2024-05
    93/100 stars

    Images

    1) Product Images from "The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways"

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    Journal: Science signaling

    doi: 10.1126/scisignal.abo4314

    (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO cells stably expressing CCR2 and the corresponding CCR2 non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of remaining CCL2 relative to CCR2 non-expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. (E) Constitutive internalization of WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and corresponding GRK KO HEK293A cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO cells stably expressing CCR2 and the corresponding CCR2 non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of remaining CCL2 relative to CCR2 non-expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. (E) Constitutive internalization of WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and corresponding GRK KO HEK293A cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) HEK293 cells stably expressing CCR2 or CCR2 with nine C-terminal serine/threonine residues mutated to alanine (CCR2-ST/9A) and non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B and C) Cells expressing CCR2 or CCR2-ST9/A transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. (D) Cells transfected with CCR2-RlucII or CCR2-ST/9A-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio for untreated controls. (E) Constitutive internalization of WT CCR2 and CCR2-ST/9A in HEK293 cells was assessed by pre-label flow cytometry. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 or SNAP-CCR2-ST/9A labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. Cells were then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.
    Figure Legend Snippet: (A) HEK293 cells stably expressing CCR2 or CCR2 with nine C-terminal serine/threonine residues mutated to alanine (CCR2-ST/9A) and non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B and C) Cells expressing CCR2 or CCR2-ST9/A transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. (D) Cells transfected with CCR2-RlucII or CCR2-ST/9A-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio for untreated controls. (E) Constitutive internalization of WT CCR2 and CCR2-ST/9A in HEK293 cells was assessed by pre-label flow cytometry. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 or SNAP-CCR2-ST/9A labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. Cells were then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of respective non-CCR2 expressing cells. (B) Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (C) Constitutive internalization of WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (D) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and β-arrestin1/2 KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. (E) THP-1 cells transduced with non-targeting gRNA (neg-gRNA) and THP-1 β-arrestin1/2 KD cells were treated with vehicle or the CCR2 inhibitor BMS681 and cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of non-scavenging control BMS681 treated cells. (F) Constitutive internalization of CCR2 in THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (G) Transwell migration of THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells at various concentrations of chemokine. Data are presented as percentages of migrated cells as compared to initial number of cells added to each well. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.
    Figure Legend Snippet: (A) WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of respective non-CCR2 expressing cells. (B) Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (C) Constitutive internalization of WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (D) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and β-arrestin1/2 KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. (E) THP-1 cells transduced with non-targeting gRNA (neg-gRNA) and THP-1 β-arrestin1/2 KD cells were treated with vehicle or the CCR2 inhibitor BMS681 and cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of non-scavenging control BMS681 treated cells. (F) Constitutive internalization of CCR2 in THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (G) Transwell migration of THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells at various concentrations of chemokine. Data are presented as percentages of migrated cells as compared to initial number of cells added to each well. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling, Transduction, Migration

    (A) Non-CCR2 expressing cells and HEK293 cells stably expressing CCR2 were transfected with two different concentrations of a dynamin dominant-negative mutant (DNM2-K44A) or left untransfected. The cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were determined by ELISA and interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B) Constitutive internalization of CCR2 was assessed by pre-label flow cytometry. HEK293 cells expressing CCR2 were transfected with pcDNA or an increasing amount of DNM2-K44A. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (C) Constitutive internalization in the presence or absence of DNM2-K44A was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 and DNM2-K44A-GFP. SNAP-CCR2 was labeled with cell impermeable SNAP-Surface Alexa Fluor 649 at 4°C for 1 h and then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being analyzed. Scale bars, 10 µm. (D) Cells transfected with CCR2-RlucII, rGFP-CAAX, and pcDNA or increasing amounts of DNM2-K44A were incubated in the absence or presence of 100 nM CCL2. CCR2 internalization was assessed by BRET. The BRET ratio changes on agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) Non-CCR2 expressing cells and HEK293 cells stably expressing CCR2 were transfected with two different concentrations of a dynamin dominant-negative mutant (DNM2-K44A) or left untransfected. The cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were determined by ELISA and interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B) Constitutive internalization of CCR2 was assessed by pre-label flow cytometry. HEK293 cells expressing CCR2 were transfected with pcDNA or an increasing amount of DNM2-K44A. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (C) Constitutive internalization in the presence or absence of DNM2-K44A was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 and DNM2-K44A-GFP. SNAP-CCR2 was labeled with cell impermeable SNAP-Surface Alexa Fluor 649 at 4°C for 1 h and then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being analyzed. Scale bars, 10 µm. (D) Cells transfected with CCR2-RlucII, rGFP-CAAX, and pcDNA or increasing amounts of DNM2-K44A were incubated in the absence or presence of 100 nM CCL2. CCR2 internalization was assessed by BRET. The BRET ratio changes on agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Expressing, Stable Transfection, Transfection, Dominant Negative Mutation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling, Incubation

    (A to C) HEK293 cells were transfected with receptor-RlucII (CCR2, ACKR3 or CXCR4) along with either rGFP-Rab4 (A), rGFP-Rab11 (B) or rGFP-Rab7 (C). Cells were stimulated at 37°C with indicated chemokine or left untreated. Data are presented as percentages of BRET values compared to untreated controls. (D to F) HEK293 cells were transfected with rGFP-CAAX and either CCR2-RlucII (D), ACKR3-RlucII (E) or CXCR4-RlucII (F). Cells were stimulated at 37°C with indicated chemokine and washed with PBS to remove chemokine or left unwashed. Data are presented as percentages of BRET values compared to non-chemokine treated controls. (G) HEK293 cells stably expressing SNAP-CCR2 were preincubated for 90 min with 10 mg/mL cycloheximide to block de novo protein synthesis and left untreated or stimulated with 100 nM CCL2 at 37°C for 45 min. The remaining SNAP-CCR2 at the surface was blocked at 4°C with SNAP-Surface block for 45 min. Cells were moved to 37 °C for 15, 45 or 75 min. Receptors were labeled with SNAP-Surface Alexa Fluor 649 at 4°C following each experimental condition and timepoint. Data are displayed as percentages of CCR2 detected relative to initial levels of surface CCR2 prior to stimulation (Start). (H, I) HEK293 cells expressing FLAG-CCR2 or FLAG-PAR1 were pretreated with 10 µg/mL cycloheximide for 90 min and left unstimulated (0 min) or stimulated with 100 nM CCL2 (to activate CCR2) or 100 μM TFLLRN (to activate PAR1) for 2 h at 37°C. Equivalent amount of cell lysates were immunoprecipitated with M2 anti-FLAG antibody and immunoblotting detection with anti-FLAG antibody. Cell lysates were analyzed for endogenous α-tubulin as controls (H). Receptor degradation was quantified and data (mean ± SEM) shown are expressed as the fraction of receptor remaining compared with untreated control cells as determined from three independent experiments (I). Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test or unpaired t test.
    Figure Legend Snippet: (A to C) HEK293 cells were transfected with receptor-RlucII (CCR2, ACKR3 or CXCR4) along with either rGFP-Rab4 (A), rGFP-Rab11 (B) or rGFP-Rab7 (C). Cells were stimulated at 37°C with indicated chemokine or left untreated. Data are presented as percentages of BRET values compared to untreated controls. (D to F) HEK293 cells were transfected with rGFP-CAAX and either CCR2-RlucII (D), ACKR3-RlucII (E) or CXCR4-RlucII (F). Cells were stimulated at 37°C with indicated chemokine and washed with PBS to remove chemokine or left unwashed. Data are presented as percentages of BRET values compared to non-chemokine treated controls. (G) HEK293 cells stably expressing SNAP-CCR2 were preincubated for 90 min with 10 mg/mL cycloheximide to block de novo protein synthesis and left untreated or stimulated with 100 nM CCL2 at 37°C for 45 min. The remaining SNAP-CCR2 at the surface was blocked at 4°C with SNAP-Surface block for 45 min. Cells were moved to 37 °C for 15, 45 or 75 min. Receptors were labeled with SNAP-Surface Alexa Fluor 649 at 4°C following each experimental condition and timepoint. Data are displayed as percentages of CCR2 detected relative to initial levels of surface CCR2 prior to stimulation (Start). (H, I) HEK293 cells expressing FLAG-CCR2 or FLAG-PAR1 were pretreated with 10 µg/mL cycloheximide for 90 min and left unstimulated (0 min) or stimulated with 100 nM CCL2 (to activate CCR2) or 100 μM TFLLRN (to activate PAR1) for 2 h at 37°C. Equivalent amount of cell lysates were immunoprecipitated with M2 anti-FLAG antibody and immunoblotting detection with anti-FLAG antibody. Cell lysates were analyzed for endogenous α-tubulin as controls (H). Receptor degradation was quantified and data (mean ± SEM) shown are expressed as the fraction of receptor remaining compared with untreated control cells as determined from three independent experiments (I). Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test or unpaired t test.

    Techniques Used: Transfection, Stable Transfection, Expressing, Blocking Assay, Labeling, Immunoprecipitation, Western Blot

    snap surface block  (New England Biolabs)


    Bioz Verified Symbol New England Biolabs is a verified supplier
    Bioz Manufacturer Symbol New England Biolabs manufactures this product  
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  • 93

    Structured Review

    New England Biolabs snap surface block
    (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared <t>to</t> <t>non-internalized</t> control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing <t>SNAP-CCR2</t> that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap surface block/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap surface block - by Bioz Stars, 2024-05
    93/100 stars

    Images

    1) Product Images from "The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways"

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    Journal: Science signaling

    doi: 10.1126/scisignal.abo4314

    (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO cells stably expressing CCR2 and the corresponding CCR2 non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of remaining CCL2 relative to CCR2 non-expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. (E) Constitutive internalization of WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and corresponding GRK KO HEK293A cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO cells stably expressing CCR2 and the corresponding CCR2 non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of remaining CCL2 relative to CCR2 non-expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. (E) Constitutive internalization of WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and corresponding GRK KO HEK293A cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) HEK293 cells stably expressing CCR2 or CCR2 with nine C-terminal serine/threonine residues mutated to alanine (CCR2-ST/9A) and non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B and C) Cells expressing CCR2 or CCR2-ST9/A transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. (D) Cells transfected with CCR2-RlucII or CCR2-ST/9A-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio for untreated controls. (E) Constitutive internalization of WT CCR2 and CCR2-ST/9A in HEK293 cells was assessed by pre-label flow cytometry. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 or SNAP-CCR2-ST/9A labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. Cells were then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.
    Figure Legend Snippet: (A) HEK293 cells stably expressing CCR2 or CCR2 with nine C-terminal serine/threonine residues mutated to alanine (CCR2-ST/9A) and non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B and C) Cells expressing CCR2 or CCR2-ST9/A transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. (D) Cells transfected with CCR2-RlucII or CCR2-ST/9A-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio for untreated controls. (E) Constitutive internalization of WT CCR2 and CCR2-ST/9A in HEK293 cells was assessed by pre-label flow cytometry. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 or SNAP-CCR2-ST/9A labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. Cells were then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of respective non-CCR2 expressing cells. (B) Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (C) Constitutive internalization of WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (D) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and β-arrestin1/2 KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. (E) THP-1 cells transduced with non-targeting gRNA (neg-gRNA) and THP-1 β-arrestin1/2 KD cells were treated with vehicle or the CCR2 inhibitor BMS681 and cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of non-scavenging control BMS681 treated cells. (F) Constitutive internalization of CCR2 in THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (G) Transwell migration of THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells at various concentrations of chemokine. Data are presented as percentages of migrated cells as compared to initial number of cells added to each well. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.
    Figure Legend Snippet: (A) WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of respective non-CCR2 expressing cells. (B) Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (C) Constitutive internalization of WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (D) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and β-arrestin1/2 KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. (E) THP-1 cells transduced with non-targeting gRNA (neg-gRNA) and THP-1 β-arrestin1/2 KD cells were treated with vehicle or the CCR2 inhibitor BMS681 and cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of non-scavenging control BMS681 treated cells. (F) Constitutive internalization of CCR2 in THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (G) Transwell migration of THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells at various concentrations of chemokine. Data are presented as percentages of migrated cells as compared to initial number of cells added to each well. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling, Transduction, Migration

    (A) Non-CCR2 expressing cells and HEK293 cells stably expressing CCR2 were transfected with two different concentrations of a dynamin dominant-negative mutant (DNM2-K44A) or left untransfected. The cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were determined by ELISA and interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B) Constitutive internalization of CCR2 was assessed by pre-label flow cytometry. HEK293 cells expressing CCR2 were transfected with pcDNA or an increasing amount of DNM2-K44A. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (C) Constitutive internalization in the presence or absence of DNM2-K44A was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 and DNM2-K44A-GFP. SNAP-CCR2 was labeled with cell impermeable SNAP-Surface Alexa Fluor 649 at 4°C for 1 h and then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being analyzed. Scale bars, 10 µm. (D) Cells transfected with CCR2-RlucII, rGFP-CAAX, and pcDNA or increasing amounts of DNM2-K44A were incubated in the absence or presence of 100 nM CCL2. CCR2 internalization was assessed by BRET. The BRET ratio changes on agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) Non-CCR2 expressing cells and HEK293 cells stably expressing CCR2 were transfected with two different concentrations of a dynamin dominant-negative mutant (DNM2-K44A) or left untransfected. The cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were determined by ELISA and interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B) Constitutive internalization of CCR2 was assessed by pre-label flow cytometry. HEK293 cells expressing CCR2 were transfected with pcDNA or an increasing amount of DNM2-K44A. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (C) Constitutive internalization in the presence or absence of DNM2-K44A was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 and DNM2-K44A-GFP. SNAP-CCR2 was labeled with cell impermeable SNAP-Surface Alexa Fluor 649 at 4°C for 1 h and then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being analyzed. Scale bars, 10 µm. (D) Cells transfected with CCR2-RlucII, rGFP-CAAX, and pcDNA or increasing amounts of DNM2-K44A were incubated in the absence or presence of 100 nM CCL2. CCR2 internalization was assessed by BRET. The BRET ratio changes on agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Expressing, Stable Transfection, Transfection, Dominant Negative Mutation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling, Incubation

    (A to C) HEK293 cells were transfected with receptor-RlucII (CCR2, ACKR3 or CXCR4) along with either rGFP-Rab4 (A), rGFP-Rab11 (B) or rGFP-Rab7 (C). Cells were stimulated at 37°C with indicated chemokine or left untreated. Data are presented as percentages of BRET values compared to untreated controls. (D to F) HEK293 cells were transfected with rGFP-CAAX and either CCR2-RlucII (D), ACKR3-RlucII (E) or CXCR4-RlucII (F). Cells were stimulated at 37°C with indicated chemokine and washed with PBS to remove chemokine or left unwashed. Data are presented as percentages of BRET values compared to non-chemokine treated controls. (G) HEK293 cells stably expressing SNAP-CCR2 were preincubated for 90 min with 10 mg/mL cycloheximide to block de novo protein synthesis and left untreated or stimulated with 100 nM CCL2 at 37°C for 45 min. The remaining SNAP-CCR2 at the surface was blocked at 4°C with SNAP-Surface block for 45 min. Cells were moved to 37 °C for 15, 45 or 75 min. Receptors were labeled with SNAP-Surface Alexa Fluor 649 at 4°C following each experimental condition and timepoint. Data are displayed as percentages of CCR2 detected relative to initial levels of surface CCR2 prior to stimulation (Start). (H, I) HEK293 cells expressing FLAG-CCR2 or FLAG-PAR1 were pretreated with 10 µg/mL cycloheximide for 90 min and left unstimulated (0 min) or stimulated with 100 nM CCL2 (to activate CCR2) or 100 μM TFLLRN (to activate PAR1) for 2 h at 37°C. Equivalent amount of cell lysates were immunoprecipitated with M2 anti-FLAG antibody and immunoblotting detection with anti-FLAG antibody. Cell lysates were analyzed for endogenous α-tubulin as controls (H). Receptor degradation was quantified and data (mean ± SEM) shown are expressed as the fraction of receptor remaining compared with untreated control cells as determined from three independent experiments (I). Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test or unpaired t test.
    Figure Legend Snippet: (A to C) HEK293 cells were transfected with receptor-RlucII (CCR2, ACKR3 or CXCR4) along with either rGFP-Rab4 (A), rGFP-Rab11 (B) or rGFP-Rab7 (C). Cells were stimulated at 37°C with indicated chemokine or left untreated. Data are presented as percentages of BRET values compared to untreated controls. (D to F) HEK293 cells were transfected with rGFP-CAAX and either CCR2-RlucII (D), ACKR3-RlucII (E) or CXCR4-RlucII (F). Cells were stimulated at 37°C with indicated chemokine and washed with PBS to remove chemokine or left unwashed. Data are presented as percentages of BRET values compared to non-chemokine treated controls. (G) HEK293 cells stably expressing SNAP-CCR2 were preincubated for 90 min with 10 mg/mL cycloheximide to block de novo protein synthesis and left untreated or stimulated with 100 nM CCL2 at 37°C for 45 min. The remaining SNAP-CCR2 at the surface was blocked at 4°C with SNAP-Surface block for 45 min. Cells were moved to 37 °C for 15, 45 or 75 min. Receptors were labeled with SNAP-Surface Alexa Fluor 649 at 4°C following each experimental condition and timepoint. Data are displayed as percentages of CCR2 detected relative to initial levels of surface CCR2 prior to stimulation (Start). (H, I) HEK293 cells expressing FLAG-CCR2 or FLAG-PAR1 were pretreated with 10 µg/mL cycloheximide for 90 min and left unstimulated (0 min) or stimulated with 100 nM CCL2 (to activate CCR2) or 100 μM TFLLRN (to activate PAR1) for 2 h at 37°C. Equivalent amount of cell lysates were immunoprecipitated with M2 anti-FLAG antibody and immunoblotting detection with anti-FLAG antibody. Cell lysates were analyzed for endogenous α-tubulin as controls (H). Receptor degradation was quantified and data (mean ± SEM) shown are expressed as the fraction of receptor remaining compared with untreated control cells as determined from three independent experiments (I). Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test or unpaired t test.

    Techniques Used: Transfection, Stable Transfection, Expressing, Blocking Assay, Labeling, Immunoprecipitation, Western Blot

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    New England Biolabs snap surface block
    (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared <t>to</t> <t>non-internalized</t> control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing <t>SNAP-CCR2</t> that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
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    1) Product Images from "The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways"

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    Journal: Science signaling

    doi: 10.1126/scisignal.abo4314

    (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO cells stably expressing CCR2 and the corresponding CCR2 non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of remaining CCL2 relative to CCR2 non-expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. (E) Constitutive internalization of WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and corresponding GRK KO HEK293A cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO cells stably expressing CCR2 and the corresponding CCR2 non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of remaining CCL2 relative to CCR2 non-expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. (E) Constitutive internalization of WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and corresponding GRK KO HEK293A cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) HEK293 cells stably expressing CCR2 or CCR2 with nine C-terminal serine/threonine residues mutated to alanine (CCR2-ST/9A) and non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B and C) Cells expressing CCR2 or CCR2-ST9/A transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. (D) Cells transfected with CCR2-RlucII or CCR2-ST/9A-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio for untreated controls. (E) Constitutive internalization of WT CCR2 and CCR2-ST/9A in HEK293 cells was assessed by pre-label flow cytometry. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 or SNAP-CCR2-ST/9A labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. Cells were then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.
    Figure Legend Snippet: (A) HEK293 cells stably expressing CCR2 or CCR2 with nine C-terminal serine/threonine residues mutated to alanine (CCR2-ST/9A) and non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B and C) Cells expressing CCR2 or CCR2-ST9/A transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. (D) Cells transfected with CCR2-RlucII or CCR2-ST/9A-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio for untreated controls. (E) Constitutive internalization of WT CCR2 and CCR2-ST/9A in HEK293 cells was assessed by pre-label flow cytometry. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 or SNAP-CCR2-ST/9A labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. Cells were then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of respective non-CCR2 expressing cells. (B) Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (C) Constitutive internalization of WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (D) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and β-arrestin1/2 KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. (E) THP-1 cells transduced with non-targeting gRNA (neg-gRNA) and THP-1 β-arrestin1/2 KD cells were treated with vehicle or the CCR2 inhibitor BMS681 and cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of non-scavenging control BMS681 treated cells. (F) Constitutive internalization of CCR2 in THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (G) Transwell migration of THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells at various concentrations of chemokine. Data are presented as percentages of migrated cells as compared to initial number of cells added to each well. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.
    Figure Legend Snippet: (A) WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of respective non-CCR2 expressing cells. (B) Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (C) Constitutive internalization of WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (D) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and β-arrestin1/2 KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. (E) THP-1 cells transduced with non-targeting gRNA (neg-gRNA) and THP-1 β-arrestin1/2 KD cells were treated with vehicle or the CCR2 inhibitor BMS681 and cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of non-scavenging control BMS681 treated cells. (F) Constitutive internalization of CCR2 in THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (G) Transwell migration of THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells at various concentrations of chemokine. Data are presented as percentages of migrated cells as compared to initial number of cells added to each well. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Techniques Used: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling, Transduction, Migration

    (A) Non-CCR2 expressing cells and HEK293 cells stably expressing CCR2 were transfected with two different concentrations of a dynamin dominant-negative mutant (DNM2-K44A) or left untransfected. The cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were determined by ELISA and interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B) Constitutive internalization of CCR2 was assessed by pre-label flow cytometry. HEK293 cells expressing CCR2 were transfected with pcDNA or an increasing amount of DNM2-K44A. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (C) Constitutive internalization in the presence or absence of DNM2-K44A was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 and DNM2-K44A-GFP. SNAP-CCR2 was labeled with cell impermeable SNAP-Surface Alexa Fluor 649 at 4°C for 1 h and then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being analyzed. Scale bars, 10 µm. (D) Cells transfected with CCR2-RlucII, rGFP-CAAX, and pcDNA or increasing amounts of DNM2-K44A were incubated in the absence or presence of 100 nM CCL2. CCR2 internalization was assessed by BRET. The BRET ratio changes on agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Figure Legend Snippet: (A) Non-CCR2 expressing cells and HEK293 cells stably expressing CCR2 were transfected with two different concentrations of a dynamin dominant-negative mutant (DNM2-K44A) or left untransfected. The cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were determined by ELISA and interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B) Constitutive internalization of CCR2 was assessed by pre-label flow cytometry. HEK293 cells expressing CCR2 were transfected with pcDNA or an increasing amount of DNM2-K44A. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (C) Constitutive internalization in the presence or absence of DNM2-K44A was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 and DNM2-K44A-GFP. SNAP-CCR2 was labeled with cell impermeable SNAP-Surface Alexa Fluor 649 at 4°C for 1 h and then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being analyzed. Scale bars, 10 µm. (D) Cells transfected with CCR2-RlucII, rGFP-CAAX, and pcDNA or increasing amounts of DNM2-K44A were incubated in the absence or presence of 100 nM CCL2. CCR2 internalization was assessed by BRET. The BRET ratio changes on agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Techniques Used: Expressing, Stable Transfection, Transfection, Dominant Negative Mutation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling, Incubation

    (A to C) HEK293 cells were transfected with receptor-RlucII (CCR2, ACKR3 or CXCR4) along with either rGFP-Rab4 (A), rGFP-Rab11 (B) or rGFP-Rab7 (C). Cells were stimulated at 37°C with indicated chemokine or left untreated. Data are presented as percentages of BRET values compared to untreated controls. (D to F) HEK293 cells were transfected with rGFP-CAAX and either CCR2-RlucII (D), ACKR3-RlucII (E) or CXCR4-RlucII (F). Cells were stimulated at 37°C with indicated chemokine and washed with PBS to remove chemokine or left unwashed. Data are presented as percentages of BRET values compared to non-chemokine treated controls. (G) HEK293 cells stably expressing SNAP-CCR2 were preincubated for 90 min with 10 mg/mL cycloheximide to block de novo protein synthesis and left untreated or stimulated with 100 nM CCL2 at 37°C for 45 min. The remaining SNAP-CCR2 at the surface was blocked at 4°C with SNAP-Surface block for 45 min. Cells were moved to 37 °C for 15, 45 or 75 min. Receptors were labeled with SNAP-Surface Alexa Fluor 649 at 4°C following each experimental condition and timepoint. Data are displayed as percentages of CCR2 detected relative to initial levels of surface CCR2 prior to stimulation (Start). (H, I) HEK293 cells expressing FLAG-CCR2 or FLAG-PAR1 were pretreated with 10 µg/mL cycloheximide for 90 min and left unstimulated (0 min) or stimulated with 100 nM CCL2 (to activate CCR2) or 100 μM TFLLRN (to activate PAR1) for 2 h at 37°C. Equivalent amount of cell lysates were immunoprecipitated with M2 anti-FLAG antibody and immunoblotting detection with anti-FLAG antibody. Cell lysates were analyzed for endogenous α-tubulin as controls (H). Receptor degradation was quantified and data (mean ± SEM) shown are expressed as the fraction of receptor remaining compared with untreated control cells as determined from three independent experiments (I). Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test or unpaired t test.
    Figure Legend Snippet: (A to C) HEK293 cells were transfected with receptor-RlucII (CCR2, ACKR3 or CXCR4) along with either rGFP-Rab4 (A), rGFP-Rab11 (B) or rGFP-Rab7 (C). Cells were stimulated at 37°C with indicated chemokine or left untreated. Data are presented as percentages of BRET values compared to untreated controls. (D to F) HEK293 cells were transfected with rGFP-CAAX and either CCR2-RlucII (D), ACKR3-RlucII (E) or CXCR4-RlucII (F). Cells were stimulated at 37°C with indicated chemokine and washed with PBS to remove chemokine or left unwashed. Data are presented as percentages of BRET values compared to non-chemokine treated controls. (G) HEK293 cells stably expressing SNAP-CCR2 were preincubated for 90 min with 10 mg/mL cycloheximide to block de novo protein synthesis and left untreated or stimulated with 100 nM CCL2 at 37°C for 45 min. The remaining SNAP-CCR2 at the surface was blocked at 4°C with SNAP-Surface block for 45 min. Cells were moved to 37 °C for 15, 45 or 75 min. Receptors were labeled with SNAP-Surface Alexa Fluor 649 at 4°C following each experimental condition and timepoint. Data are displayed as percentages of CCR2 detected relative to initial levels of surface CCR2 prior to stimulation (Start). (H, I) HEK293 cells expressing FLAG-CCR2 or FLAG-PAR1 were pretreated with 10 µg/mL cycloheximide for 90 min and left unstimulated (0 min) or stimulated with 100 nM CCL2 (to activate CCR2) or 100 μM TFLLRN (to activate PAR1) for 2 h at 37°C. Equivalent amount of cell lysates were immunoprecipitated with M2 anti-FLAG antibody and immunoblotting detection with anti-FLAG antibody. Cell lysates were analyzed for endogenous α-tubulin as controls (H). Receptor degradation was quantified and data (mean ± SEM) shown are expressed as the fraction of receptor remaining compared with untreated control cells as determined from three independent experiments (I). Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test or unpaired t test.

    Techniques Used: Transfection, Stable Transfection, Expressing, Blocking Assay, Labeling, Immunoprecipitation, Western Blot

    snap surface block  (New England Biolabs)


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    New England Biolabs snap surface block
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs snap substrates
    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with <t>SNAP</t> surface <t>dye</t> <t>Alexa</t> Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Snap Substrates, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion"

    Article Title: Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2022.101541

    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Figure Legend Snippet: Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).

    Techniques Used: Construct, Clone Assay, Western Blot, Incubation, Flow Cytometry, Labeling

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    New England Biolabs snap surface block
    (A-B”) Confocal images of third instar larva imaginal wing discs expressing UAS <t>SNAP-smo</t> wt (driven by apGal4) in the dorsal compartment and labeled with a SNAP cell-permeable fluorescent substrate (A, B and red in the merge images A”, B”) and DLG (A’ and green in A’’ and B’’). (B’) Total SNAP-SMO in false colour. (A-A”): XZ section, (B-B”): XZ projection. (C-D’’) Confocal images of imaginal discs expressing UAS SNAP-smo wt and labeled with a non-liposoluble fluorescent SNAP substrate followed by a cell-permeable fluorescent SNAP substrate. C-C” XZ section, D-D” XZ projection. Here and in the to : XZ sections are antero-posterior Z sections in the dorsal compartment. The disc images are oriented anterior to the left and apical up. The dotted vertical line represents the A/P boundary (determined by CI staining). The Z projection images correspond to an average intensity projection of 8 sections. The scale bars represent 50 μm for XY images and 10 μm for XZ images. (E-E”) Quantification of SNAP-SMO localization in the XZ sections. (E) Density (mean intensity) of total (pale <t>grey),</t> <t>intracellular</t> (Intra, red) and surface (Surf, dark grey) SNAP-SMO in two different regions of the wing imaginal disc: far anterior (FA) region (empty bars here and in - ) where no HH is present and posterior (P) region (bars with diagonal lines here and in - . (E’ and E”) Distribution (intensity reported to the intensity of the whole column taken for quantification) of Total SNAP-SMO (E’) or of surface SNAP-SMO (E’’) in the apical (light blue), lateral (medium blue) and basal (dark blue) domains in the FA or P regions. Total SNAP-SMO: N=9 discs, for Intra and Surf: N=12 discs. The error bars represent the S.D. and the statistical analysis was performed using paired t-test for the mean intensities and Wilcoxon matched-pairs signed rank tests for the relative intensities. (F-F”) Confocal images of imaginal discs expressing UAS SNAP-smo WT , labeled with a non-liposoluble fluorescent SNAP substrate (F, G green in the merge images F”and G”) and immunolabeled for unprocessed CI (F’, G’, blue in F”and G”). XY images in (F to F’’) with anterior left and dorsal up. XZ images in (G to G”).The disc is divided in four regions (separated by dotted lines) along the anterior posterior axis based on CI staining: the posterior (green), and three anterior regions: CI-A (low levels of full-length activated CI, in purple), CI-F (higher levels of CI full-length, in pink) and CI-R (CI repressor not detected by the anti CI antibody used here, in red). See also Figure S1E. (H) Graph showing the relative intensity of surface SNAP-SMO in the apical, lateral and basal domains in CI-R, CI-FL, CI-A and P regions. N= 33. For all Figures, the precise genotypes are given in the Table S1, the statistical tests used and the p-values are indicated in the Table S2. See also Figure S1.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "High Hedgehog signaling is transduced by a multikinase-dependent switch controling the apico-basal distribution of the GPCR Smoothened"

    Article Title: High Hedgehog signaling is transduced by a multikinase-dependent switch controling the apico-basal distribution of the GPCR Smoothened

    Journal: bioRxiv

    doi: 10.1101/2022.01.19.476759

    (A-B”) Confocal images of third instar larva imaginal wing discs expressing UAS SNAP-smo wt (driven by apGal4) in the dorsal compartment and labeled with a SNAP cell-permeable fluorescent substrate (A, B and red in the merge images A”, B”) and DLG (A’ and green in A’’ and B’’). (B’) Total SNAP-SMO in false colour. (A-A”): XZ section, (B-B”): XZ projection. (C-D’’) Confocal images of imaginal discs expressing UAS SNAP-smo wt and labeled with a non-liposoluble fluorescent SNAP substrate followed by a cell-permeable fluorescent SNAP substrate. C-C” XZ section, D-D” XZ projection. Here and in the to : XZ sections are antero-posterior Z sections in the dorsal compartment. The disc images are oriented anterior to the left and apical up. The dotted vertical line represents the A/P boundary (determined by CI staining). The Z projection images correspond to an average intensity projection of 8 sections. The scale bars represent 50 μm for XY images and 10 μm for XZ images. (E-E”) Quantification of SNAP-SMO localization in the XZ sections. (E) Density (mean intensity) of total (pale grey), intracellular (Intra, red) and surface (Surf, dark grey) SNAP-SMO in two different regions of the wing imaginal disc: far anterior (FA) region (empty bars here and in - ) where no HH is present and posterior (P) region (bars with diagonal lines here and in - . (E’ and E”) Distribution (intensity reported to the intensity of the whole column taken for quantification) of Total SNAP-SMO (E’) or of surface SNAP-SMO (E’’) in the apical (light blue), lateral (medium blue) and basal (dark blue) domains in the FA or P regions. Total SNAP-SMO: N=9 discs, for Intra and Surf: N=12 discs. The error bars represent the S.D. and the statistical analysis was performed using paired t-test for the mean intensities and Wilcoxon matched-pairs signed rank tests for the relative intensities. (F-F”) Confocal images of imaginal discs expressing UAS SNAP-smo WT , labeled with a non-liposoluble fluorescent SNAP substrate (F, G green in the merge images F”and G”) and immunolabeled for unprocessed CI (F’, G’, blue in F”and G”). XY images in (F to F’’) with anterior left and dorsal up. XZ images in (G to G”).The disc is divided in four regions (separated by dotted lines) along the anterior posterior axis based on CI staining: the posterior (green), and three anterior regions: CI-A (low levels of full-length activated CI, in purple), CI-F (higher levels of CI full-length, in pink) and CI-R (CI repressor not detected by the anti CI antibody used here, in red). See also Figure S1E. (H) Graph showing the relative intensity of surface SNAP-SMO in the apical, lateral and basal domains in CI-R, CI-FL, CI-A and P regions. N= 33. For all Figures, the precise genotypes are given in the Table S1, the statistical tests used and the p-values are indicated in the Table S2. See also Figure S1.
    Figure Legend Snippet: (A-B”) Confocal images of third instar larva imaginal wing discs expressing UAS SNAP-smo wt (driven by apGal4) in the dorsal compartment and labeled with a SNAP cell-permeable fluorescent substrate (A, B and red in the merge images A”, B”) and DLG (A’ and green in A’’ and B’’). (B’) Total SNAP-SMO in false colour. (A-A”): XZ section, (B-B”): XZ projection. (C-D’’) Confocal images of imaginal discs expressing UAS SNAP-smo wt and labeled with a non-liposoluble fluorescent SNAP substrate followed by a cell-permeable fluorescent SNAP substrate. C-C” XZ section, D-D” XZ projection. Here and in the to : XZ sections are antero-posterior Z sections in the dorsal compartment. The disc images are oriented anterior to the left and apical up. The dotted vertical line represents the A/P boundary (determined by CI staining). The Z projection images correspond to an average intensity projection of 8 sections. The scale bars represent 50 μm for XY images and 10 μm for XZ images. (E-E”) Quantification of SNAP-SMO localization in the XZ sections. (E) Density (mean intensity) of total (pale grey), intracellular (Intra, red) and surface (Surf, dark grey) SNAP-SMO in two different regions of the wing imaginal disc: far anterior (FA) region (empty bars here and in - ) where no HH is present and posterior (P) region (bars with diagonal lines here and in - . (E’ and E”) Distribution (intensity reported to the intensity of the whole column taken for quantification) of Total SNAP-SMO (E’) or of surface SNAP-SMO (E’’) in the apical (light blue), lateral (medium blue) and basal (dark blue) domains in the FA or P regions. Total SNAP-SMO: N=9 discs, for Intra and Surf: N=12 discs. The error bars represent the S.D. and the statistical analysis was performed using paired t-test for the mean intensities and Wilcoxon matched-pairs signed rank tests for the relative intensities. (F-F”) Confocal images of imaginal discs expressing UAS SNAP-smo WT , labeled with a non-liposoluble fluorescent SNAP substrate (F, G green in the merge images F”and G”) and immunolabeled for unprocessed CI (F’, G’, blue in F”and G”). XY images in (F to F’’) with anterior left and dorsal up. XZ images in (G to G”).The disc is divided in four regions (separated by dotted lines) along the anterior posterior axis based on CI staining: the posterior (green), and three anterior regions: CI-A (low levels of full-length activated CI, in purple), CI-F (higher levels of CI full-length, in pink) and CI-R (CI repressor not detected by the anti CI antibody used here, in red). See also Figure S1E. (H) Graph showing the relative intensity of surface SNAP-SMO in the apical, lateral and basal domains in CI-R, CI-FL, CI-A and P regions. N= 33. For all Figures, the precise genotypes are given in the Table S1, the statistical tests used and the p-values are indicated in the Table S2. See also Figure S1.

    Techniques Used: Expressing, Labeling, Staining, Immunolabeling

    snap surface block  (New England Biolabs)


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    New England Biolabs snap surface block
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    snap surface block 480  (New England Biolabs)


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    New England Biolabs snap surface block
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    New England Biolabs snap surface block
    CBG <t>and</t> <t>SBG-conjugated</t> SiR are membrane-impermeable and enable specific targeting of surface proteins. (a) Application of permeable BG-SiR to N-terminally <t>SNAP-fused</t> GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. (b) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. (c) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. (d and e) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted (d) or cytosol-targeted (e) SNAP-tags. Labelling concentration was 500 nM for all compounds. (f and g) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labelling restricted to the surface only with SBG-SiR. (h) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 μm.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates"

    Article Title: Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

    Journal: Chemical Science

    doi: 10.1039/d0sc02794d

    CBG and SBG-conjugated SiR are membrane-impermeable and enable specific targeting of surface proteins. (a) Application of permeable BG-SiR to N-terminally SNAP-fused GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. (b) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. (c) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. (d and e) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted (d) or cytosol-targeted (e) SNAP-tags. Labelling concentration was 500 nM for all compounds. (f and g) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labelling restricted to the surface only with SBG-SiR. (h) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 μm.
    Figure Legend Snippet: CBG and SBG-conjugated SiR are membrane-impermeable and enable specific targeting of surface proteins. (a) Application of permeable BG-SiR to N-terminally SNAP-fused GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. (b) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. (c) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. (d and e) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted (d) or cytosol-targeted (e) SNAP-tags. Labelling concentration was 500 nM for all compounds. (f and g) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labelling restricted to the surface only with SBG-SiR. (h) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 μm.

    Techniques Used: Concentration Assay

    SBG-conjugated fluorophores across the visible spectrum enable surface-specific SNAP labelling and nanoscopic imaging of surface receptors. (a–d) SBG conjugation enables surface targeting of Oregon Green (a), TMR (b), JF 549 (c), and JF 646 (d). All fluorophores label nuclear SNAP-tags when conjugated BG but not SBG, for which they show cleaner surface targeting of SNAP-mGluR2. (e and f) Confocal and superresolution STED nanoscopy of mGluR2 using BG-JF 646 (e) and SBG-JF 646 (f) shows clear isolation of the membrane population only using the impermeable SBG probe. Labelling concentration was 500 nM for all compounds. Data is represented as mean ± SEM. Scale bars are 20 μm.
    Figure Legend Snippet: SBG-conjugated fluorophores across the visible spectrum enable surface-specific SNAP labelling and nanoscopic imaging of surface receptors. (a–d) SBG conjugation enables surface targeting of Oregon Green (a), TMR (b), JF 549 (c), and JF 646 (d). All fluorophores label nuclear SNAP-tags when conjugated BG but not SBG, for which they show cleaner surface targeting of SNAP-mGluR2. (e and f) Confocal and superresolution STED nanoscopy of mGluR2 using BG-JF 646 (e) and SBG-JF 646 (f) shows clear isolation of the membrane population only using the impermeable SBG probe. Labelling concentration was 500 nM for all compounds. Data is represented as mean ± SEM. Scale bars are 20 μm.

    Techniques Used: Imaging, Conjugation Assay, Isolation, Concentration Assay

    In vivo labelling of a SNAP-tagged receptor with SBG-conjugated fluorophores produces less background and more spread. (a) Schematic showing AAV-mediated expression of SNAP-mGluR2 in the medial prefrontal cortex (mPFC) of mice, followed by SBG-JF 549 or BG-JF 549 dye injection and slice preparation 8 weeks later. (b and c) Representative images showing fluorescence in slices from SNAP-mGluR2 expressing mice following injection of BG (b) or SBG (c) fluorophores. (d) SBG-JF 549 shows broader spread throughout the cortex compared to BG-JF 549 . * indicates statistical significance (unpaired t -test, p = 0.04). (e and f) Representative images showing fluorescence in control slices following injection of BG (e) or SBG (f) fluorophores. (g) Larger background signals are observed for BG-conjugated dye. * indicates statistical significance (unpaired t -test, p = 0.007). Data is represented as mean ± SEM and comes from n = 3 mice for each condition. Labelling concentration was 1 μM. Scale bars are 150 μm.
    Figure Legend Snippet: In vivo labelling of a SNAP-tagged receptor with SBG-conjugated fluorophores produces less background and more spread. (a) Schematic showing AAV-mediated expression of SNAP-mGluR2 in the medial prefrontal cortex (mPFC) of mice, followed by SBG-JF 549 or BG-JF 549 dye injection and slice preparation 8 weeks later. (b and c) Representative images showing fluorescence in slices from SNAP-mGluR2 expressing mice following injection of BG (b) or SBG (c) fluorophores. (d) SBG-JF 549 shows broader spread throughout the cortex compared to BG-JF 549 . * indicates statistical significance (unpaired t -test, p = 0.04). (e and f) Representative images showing fluorescence in control slices following injection of BG (e) or SBG (f) fluorophores. (g) Larger background signals are observed for BG-conjugated dye. * indicates statistical significance (unpaired t -test, p = 0.007). Data is represented as mean ± SEM and comes from n = 3 mice for each condition. Labelling concentration was 1 μM. Scale bars are 150 μm.

    Techniques Used: In Vivo, Expressing, Injection, Slice Preparation, Fluorescence, Concentration Assay

    BG and SBG-conjugated fluorophores enable SiMPull analysis of isolated surface or intracellular receptor populations. (a) Representative images showing labelling of either surface (top) or intracellular (bottom) SNAP-mGluR2 with SBG- or SNAP-Surface® Block followed by BG-JF 549 , respectively. (b) Schematic showing single molecule pulldown configuration where an anti-HA antibody is used to isolate a sparse surface of SNAP-tagged mGluR2 following fluorophore labelling. (c) Representative image of single molecules for SNAP-mGluR2, with representative bleaching traces for a 1-step and 2-step example (bottom). Note: >95% of spots bleached in either 1 or 2-steps. (d) Summary of the proportion of 2-step bleaching steps for each labelling configuration. Each point represents one independent movie and bars show mean ± SEM. * indicates statistical significance (unpaired t test, p = 0.0005). (e–h) Same as (a–d) but with SNAP-β2AR. * indicates statistical significance (unpaired t test, p = 0.008). Scale bars are 10 μm. (i) Representative images showing 2-colour labelling of either surface SNAP-mGluR2 exclusively (top) or surface and intracellular SNAP-mGluR2 (bottom). All fluorophores were applied at 1 μM. (j) Representative images of single molecules in two different colours, with co-localized spots circled. (k) Summary of the proportion of total spots that are co-localized between the two colours. Each point represents one independent movie and bars show mean ± SEM.
    Figure Legend Snippet: BG and SBG-conjugated fluorophores enable SiMPull analysis of isolated surface or intracellular receptor populations. (a) Representative images showing labelling of either surface (top) or intracellular (bottom) SNAP-mGluR2 with SBG- or SNAP-Surface® Block followed by BG-JF 549 , respectively. (b) Schematic showing single molecule pulldown configuration where an anti-HA antibody is used to isolate a sparse surface of SNAP-tagged mGluR2 following fluorophore labelling. (c) Representative image of single molecules for SNAP-mGluR2, with representative bleaching traces for a 1-step and 2-step example (bottom). Note: >95% of spots bleached in either 1 or 2-steps. (d) Summary of the proportion of 2-step bleaching steps for each labelling configuration. Each point represents one independent movie and bars show mean ± SEM. * indicates statistical significance (unpaired t test, p = 0.0005). (e–h) Same as (a–d) but with SNAP-β2AR. * indicates statistical significance (unpaired t test, p = 0.008). Scale bars are 10 μm. (i) Representative images showing 2-colour labelling of either surface SNAP-mGluR2 exclusively (top) or surface and intracellular SNAP-mGluR2 (bottom). All fluorophores were applied at 1 μM. (j) Representative images of single molecules in two different colours, with co-localized spots circled. (k) Summary of the proportion of total spots that are co-localized between the two colours. Each point represents one independent movie and bars show mean ± SEM.

    Techniques Used: Isolation, Blocking Assay

    snap surface block  (New England Biolabs)


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    New England Biolabs snap surface block
    a ) Application of permeable BG-SiR to N-terminally <t>SNAP-fused</t> GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. b ) Application of <t>impermeable</t> <t>SBG-SiR</t> should lead solely to labelling of extracellular tags with reduced background. c ) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. d, e ) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted ( d ) or cytosol-targeted ( e ) SNAP-tags. f, g ) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labeling restricted to the surface only with SBG-SiR. h ) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 µm.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates"

    Article Title: Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.924829

    a ) Application of permeable BG-SiR to N-terminally SNAP-fused GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. b ) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. c ) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. d, e ) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted ( d ) or cytosol-targeted ( e ) SNAP-tags. f, g ) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labeling restricted to the surface only with SBG-SiR. h ) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 µm.
    Figure Legend Snippet: a ) Application of permeable BG-SiR to N-terminally SNAP-fused GPCRs leads to extracellular labelling of surface receptors and background signals due to labelling of intracellular pools and non-specific dye accumulation. b ) Application of impermeable SBG-SiR should lead solely to labelling of extracellular tags with reduced background. c ) Chemical structures of BG-SiR, CBG-SiR and SBG-SiR. d, e ) BG-SiR, but not CBG-SiR and SBG-SiR, labels nucleus-targeted ( d ) or cytosol-targeted ( e ) SNAP-tags. f, g ) Concentration-dependent labelling of SNAP-mGluR2 leads to intracellular background signals using BG-SiR, which is absent using SBG-SiR. Line scans, right, demonstrate that labeling restricted to the surface only with SBG-SiR. h ) Untransfected cells show background signals from BG-SiR but not from SBG-SiR. Scale bars are 20 µm.

    Techniques Used: Concentration Assay, Labeling

    a-d ) SBG conjugation enables surface targeting of Oregon Green ( a ), TMR ( b ), JF 549 ( c ), and JF 646 ( d ). All fluorophores are able to label nuclear SNAP-tags when conjugated BG but not SBG and show cleaner surface targeting of SNAP-mGluR2. e, f ) Confocal and superresolution STED nanoscopy of mGluR2 using BG-JF 646 ( e ) and SBG-JF 646 ( f ) shows clear isolation of the membrane population only using the impermeable SBG probe. Data is represented as mean ± SEM. Scale bars are 20 µm.
    Figure Legend Snippet: a-d ) SBG conjugation enables surface targeting of Oregon Green ( a ), TMR ( b ), JF 549 ( c ), and JF 646 ( d ). All fluorophores are able to label nuclear SNAP-tags when conjugated BG but not SBG and show cleaner surface targeting of SNAP-mGluR2. e, f ) Confocal and superresolution STED nanoscopy of mGluR2 using BG-JF 646 ( e ) and SBG-JF 646 ( f ) shows clear isolation of the membrane population only using the impermeable SBG probe. Data is represented as mean ± SEM. Scale bars are 20 µm.

    Techniques Used: Conjugation Assay, Isolation

    a ) Schematic showing AAV-mediated expression of SNAP-mGluR2 in the medial prefrontal cortex of mice, followed by SBG-JF 549 or BG-JF 549 dye injection and slice preparation 8 weeks later. b-c ) Representative images showing fluorescence in slices from SNAP-mGluR2 expressing mice following injection of BG ( b ) or SBG ( c ) fluorophores. d ) SBG-JF 549 shows broader spread throughout the cortex compared to BG-JF 549 . * indicates statistical significance (unpaired t-test, p=0.04). e, f ) Representative images showing fluorescence in control slices following injection of BG ( e ) or SBG ( f ) fluorophores. g ) Larger background signals are observed for BG-conjugated dye. * indicates statistical significance (unpaired t-test, p=0.007). Data is represented as mean ± SEM and comes from n=3 mice for each condition. Scale bars are 150 µm.
    Figure Legend Snippet: a ) Schematic showing AAV-mediated expression of SNAP-mGluR2 in the medial prefrontal cortex of mice, followed by SBG-JF 549 or BG-JF 549 dye injection and slice preparation 8 weeks later. b-c ) Representative images showing fluorescence in slices from SNAP-mGluR2 expressing mice following injection of BG ( b ) or SBG ( c ) fluorophores. d ) SBG-JF 549 shows broader spread throughout the cortex compared to BG-JF 549 . * indicates statistical significance (unpaired t-test, p=0.04). e, f ) Representative images showing fluorescence in control slices following injection of BG ( e ) or SBG ( f ) fluorophores. g ) Larger background signals are observed for BG-conjugated dye. * indicates statistical significance (unpaired t-test, p=0.007). Data is represented as mean ± SEM and comes from n=3 mice for each condition. Scale bars are 150 µm.

    Techniques Used: Expressing, Injection, Slice Preparation, Fluorescence

    a ) Representative images showing labeling of either surface (top) or intracellular (bottom) SNAP-mGluR2 with SBG- or BG-JF 549 , respectively. b ) Schematic showing single molecule pulldown configuration where an anti-HA antibody is used to isolate a sparse surface of SNAP-tagged mGluR2 following fluorophore labeling. c ) Representative image of single molecules for SNAP-mGluR2, with representative bleaching traces for a 1-step and 2-step example (bottom). Note: >95% of spots bleached in either 1 or 2-steps. d ) Summary of the proportion of 2-step bleaching steps for each labeling configuration. Each point represents one independent movie and bars show mean ± SEM. * indicates statistical significance (unpaired t test, p=0.0005). e-h ) Same as a-d but with SNAP-ß2AR. * indicates statistical significance (unpaired t test, p=0.008). Scale bars are 10 µm.
    Figure Legend Snippet: a ) Representative images showing labeling of either surface (top) or intracellular (bottom) SNAP-mGluR2 with SBG- or BG-JF 549 , respectively. b ) Schematic showing single molecule pulldown configuration where an anti-HA antibody is used to isolate a sparse surface of SNAP-tagged mGluR2 following fluorophore labeling. c ) Representative image of single molecules for SNAP-mGluR2, with representative bleaching traces for a 1-step and 2-step example (bottom). Note: >95% of spots bleached in either 1 or 2-steps. d ) Summary of the proportion of 2-step bleaching steps for each labeling configuration. Each point represents one independent movie and bars show mean ± SEM. * indicates statistical significance (unpaired t test, p=0.0005). e-h ) Same as a-d but with SNAP-ß2AR. * indicates statistical significance (unpaired t test, p=0.008). Scale bars are 10 µm.

    Techniques Used: Labeling

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    New England Biolabs snap surface block
    (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared <t>to</t> <t>non-internalized</t> control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing <t>SNAP-CCR2</t> that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.
    Snap Surface Block, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs snap substrates
    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with <t>SNAP</t> surface <t>dye</t> <t>Alexa</t> Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Snap Substrates, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs snap surface block 480
    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with <t>SNAP</t> surface <t>dye</t> <t>Alexa</t> Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).
    Snap Surface Block 480, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snap surface block 480/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    snap surface block 480 - by Bioz Stars, 2024-05
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    Image Search Results


    (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Journal: Science signaling

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    doi: 10.1126/scisignal.abo4314

    Figure Lengend Snippet: (A) WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were measured by ELISA and interpolated from CCL2 standards, and are presented as percentages of remaining CCL2 relative to non-CCR2 expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (E) Constitutive internalization of WT, Gαi KO and Gα_all KO HEK293 cells stably expressing CCR2 were assessed by pre-label flow cytometry. Data are presented as the percentage of surface receptor remaining compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT, Gαi KO and Gα_all KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Article Snippet: Subsequently, cells were washed and remaining non-internalized SNAP-CCR2 at the cell surface was blocked at 4°C with SNAP-Surface block (New England Biolabs) for 45 min.

    Techniques: Stable Transfection, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO cells stably expressing CCR2 and the corresponding CCR2 non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of remaining CCL2 relative to CCR2 non-expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. (E) Constitutive internalization of WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and corresponding GRK KO HEK293A cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Journal: Science signaling

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    doi: 10.1126/scisignal.abo4314

    Figure Lengend Snippet: (A) WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO cells stably expressing CCR2 and the corresponding CCR2 non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of remaining CCL2 relative to CCR2 non-expressing cells. (B and C) Cells transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. Data are presented as percentages of BRET values for untreated controls. (D) CCR2 internalization assessed by BRET. Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. (E) Constitutive internalization of WT HEK293A, HEK293A GRK2/3 KO, HEK293A GRK5/6 KO and HEK293A GRK2/3/5/6 KO stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and corresponding GRK KO HEK293A cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Article Snippet: Subsequently, cells were washed and remaining non-internalized SNAP-CCR2 at the cell surface was blocked at 4°C with SNAP-Surface block (New England Biolabs) for 45 min.

    Techniques: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) HEK293 cells stably expressing CCR2 or CCR2 with nine C-terminal serine/threonine residues mutated to alanine (CCR2-ST/9A) and non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B and C) Cells expressing CCR2 or CCR2-ST9/A transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. (D) Cells transfected with CCR2-RlucII or CCR2-ST/9A-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio for untreated controls. (E) Constitutive internalization of WT CCR2 and CCR2-ST/9A in HEK293 cells was assessed by pre-label flow cytometry. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 or SNAP-CCR2-ST/9A labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. Cells were then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Journal: Science signaling

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    doi: 10.1126/scisignal.abo4314

    Figure Lengend Snippet: (A) HEK293 cells stably expressing CCR2 or CCR2 with nine C-terminal serine/threonine residues mutated to alanine (CCR2-ST/9A) and non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B and C) Cells expressing CCR2 or CCR2-ST9/A transfected with β-arrestin1- or β-arrestin2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. β-arrestin recruitment was assessed by ebBRET. (D) Cells transfected with CCR2-RlucII or CCR2-ST/9A-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio for untreated controls. (E) Constitutive internalization of WT CCR2 and CCR2-ST/9A in HEK293 cells was assessed by pre-label flow cytometry. (F) Constitutive internalization was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 or SNAP-CCR2-ST/9A labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. Cells were then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Article Snippet: Subsequently, cells were washed and remaining non-internalized SNAP-CCR2 at the cell surface was blocked at 4°C with SNAP-Surface block (New England Biolabs) for 45 min.

    Techniques: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling

    (A) WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of respective non-CCR2 expressing cells. (B) Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (C) Constitutive internalization of WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (D) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and β-arrestin1/2 KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. (E) THP-1 cells transduced with non-targeting gRNA (neg-gRNA) and THP-1 β-arrestin1/2 KD cells were treated with vehicle or the CCR2 inhibitor BMS681 and cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of non-scavenging control BMS681 treated cells. (F) Constitutive internalization of CCR2 in THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (G) Transwell migration of THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells at various concentrations of chemokine. Data are presented as percentages of migrated cells as compared to initial number of cells added to each well. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Journal: Science signaling

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    doi: 10.1126/scisignal.abo4314

    Figure Lengend Snippet: (A) WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 and respective non-expressing cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of respective non-CCR2 expressing cells. (B) Cells transfected with CCR2-RlucII and rGFP-CAAX were stimulated with 100 nM CCL2 or left untreated. CCR2 internalization was assessed by BRET. The BRET ratio changes upon agonist treatment are expressed as percentages of the BRET ratio observed in untreated controls. (C) Constitutive internalization of WT HEK293 and β-arrestin1/2 KO HEK293 cells stably expressing CCR2 was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (D) Constitutive internalization was visualized by fluorescence confocal microscopy in WT and β-arrestin1/2 KO HEK293 cells expressing SNAP-CCR2 that was labeled with cell impermeable SNAP-Surface Alexa Fluor 488 at 4°C for 1 h. The cells were then held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being imaged. Images are representative of three independent experiments. Scale bars, 10 µm. (E) THP-1 cells transduced with non-targeting gRNA (neg-gRNA) and THP-1 β-arrestin1/2 KD cells were treated with vehicle or the CCR2 inhibitor BMS681 and cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were interpolated from CCL2 standards and are presented as percentages of non-scavenging control BMS681 treated cells. (F) Constitutive internalization of CCR2 in THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells was assessed by pre-label flow cytometry. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (G) Transwell migration of THP-1 neg-gRNA and β-arrestin1/2 KD THP-1 cells at various concentrations of chemokine. Data are presented as percentages of migrated cells as compared to initial number of cells added to each well. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by unpaired t test.

    Article Snippet: Subsequently, cells were washed and remaining non-internalized SNAP-CCR2 at the cell surface was blocked at 4°C with SNAP-Surface block (New England Biolabs) for 45 min.

    Techniques: Stable Transfection, Expressing, Cell Culture, Transfection, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling, Transduction, Migration

    (A) Non-CCR2 expressing cells and HEK293 cells stably expressing CCR2 were transfected with two different concentrations of a dynamin dominant-negative mutant (DNM2-K44A) or left untransfected. The cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were determined by ELISA and interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B) Constitutive internalization of CCR2 was assessed by pre-label flow cytometry. HEK293 cells expressing CCR2 were transfected with pcDNA or an increasing amount of DNM2-K44A. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (C) Constitutive internalization in the presence or absence of DNM2-K44A was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 and DNM2-K44A-GFP. SNAP-CCR2 was labeled with cell impermeable SNAP-Surface Alexa Fluor 649 at 4°C for 1 h and then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being analyzed. Scale bars, 10 µm. (D) Cells transfected with CCR2-RlucII, rGFP-CAAX, and pcDNA or increasing amounts of DNM2-K44A were incubated in the absence or presence of 100 nM CCL2. CCR2 internalization was assessed by BRET. The BRET ratio changes on agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Journal: Science signaling

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    doi: 10.1126/scisignal.abo4314

    Figure Lengend Snippet: (A) Non-CCR2 expressing cells and HEK293 cells stably expressing CCR2 were transfected with two different concentrations of a dynamin dominant-negative mutant (DNM2-K44A) or left untransfected. The cells were cultured in media containing 5 nM CCL2 for 16 h. The remaining levels of CCL2 were determined by ELISA and interpolated from CCL2 standards and are presented as percentages of the respective non-CCR2 expressing cells. (B) Constitutive internalization of CCR2 was assessed by pre-label flow cytometry. HEK293 cells expressing CCR2 were transfected with pcDNA or an increasing amount of DNM2-K44A. Data are presented as percentages of surface receptor remaining as compared to non-internalized control. (C) Constitutive internalization in the presence or absence of DNM2-K44A was visualized by fluorescence confocal microscopy in HEK293 cells expressing SNAP-CCR2 and DNM2-K44A-GFP. SNAP-CCR2 was labeled with cell impermeable SNAP-Surface Alexa Fluor 649 at 4°C for 1 h and then either held at 4°C for 45 min (top panel) or transferred to 37°C for 45 min (bottom panel) before being analyzed. Scale bars, 10 µm. (D) Cells transfected with CCR2-RlucII, rGFP-CAAX, and pcDNA or increasing amounts of DNM2-K44A were incubated in the absence or presence of 100 nM CCL2. CCR2 internalization was assessed by BRET. The BRET ratio changes on agonist treatment are expressed as percentages of the BRET ratio observed in the untreated controls. Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test.

    Article Snippet: Subsequently, cells were washed and remaining non-internalized SNAP-CCR2 at the cell surface was blocked at 4°C with SNAP-Surface block (New England Biolabs) for 45 min.

    Techniques: Expressing, Stable Transfection, Transfection, Dominant Negative Mutation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Confocal Microscopy, Labeling, Incubation

    (A to C) HEK293 cells were transfected with receptor-RlucII (CCR2, ACKR3 or CXCR4) along with either rGFP-Rab4 (A), rGFP-Rab11 (B) or rGFP-Rab7 (C). Cells were stimulated at 37°C with indicated chemokine or left untreated. Data are presented as percentages of BRET values compared to untreated controls. (D to F) HEK293 cells were transfected with rGFP-CAAX and either CCR2-RlucII (D), ACKR3-RlucII (E) or CXCR4-RlucII (F). Cells were stimulated at 37°C with indicated chemokine and washed with PBS to remove chemokine or left unwashed. Data are presented as percentages of BRET values compared to non-chemokine treated controls. (G) HEK293 cells stably expressing SNAP-CCR2 were preincubated for 90 min with 10 mg/mL cycloheximide to block de novo protein synthesis and left untreated or stimulated with 100 nM CCL2 at 37°C for 45 min. The remaining SNAP-CCR2 at the surface was blocked at 4°C with SNAP-Surface block for 45 min. Cells were moved to 37 °C for 15, 45 or 75 min. Receptors were labeled with SNAP-Surface Alexa Fluor 649 at 4°C following each experimental condition and timepoint. Data are displayed as percentages of CCR2 detected relative to initial levels of surface CCR2 prior to stimulation (Start). (H, I) HEK293 cells expressing FLAG-CCR2 or FLAG-PAR1 were pretreated with 10 µg/mL cycloheximide for 90 min and left unstimulated (0 min) or stimulated with 100 nM CCL2 (to activate CCR2) or 100 μM TFLLRN (to activate PAR1) for 2 h at 37°C. Equivalent amount of cell lysates were immunoprecipitated with M2 anti-FLAG antibody and immunoblotting detection with anti-FLAG antibody. Cell lysates were analyzed for endogenous α-tubulin as controls (H). Receptor degradation was quantified and data (mean ± SEM) shown are expressed as the fraction of receptor remaining compared with untreated control cells as determined from three independent experiments (I). Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test or unpaired t test.

    Journal: Science signaling

    Article Title: The dual function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct pathways

    doi: 10.1126/scisignal.abo4314

    Figure Lengend Snippet: (A to C) HEK293 cells were transfected with receptor-RlucII (CCR2, ACKR3 or CXCR4) along with either rGFP-Rab4 (A), rGFP-Rab11 (B) or rGFP-Rab7 (C). Cells were stimulated at 37°C with indicated chemokine or left untreated. Data are presented as percentages of BRET values compared to untreated controls. (D to F) HEK293 cells were transfected with rGFP-CAAX and either CCR2-RlucII (D), ACKR3-RlucII (E) or CXCR4-RlucII (F). Cells were stimulated at 37°C with indicated chemokine and washed with PBS to remove chemokine or left unwashed. Data are presented as percentages of BRET values compared to non-chemokine treated controls. (G) HEK293 cells stably expressing SNAP-CCR2 were preincubated for 90 min with 10 mg/mL cycloheximide to block de novo protein synthesis and left untreated or stimulated with 100 nM CCL2 at 37°C for 45 min. The remaining SNAP-CCR2 at the surface was blocked at 4°C with SNAP-Surface block for 45 min. Cells were moved to 37 °C for 15, 45 or 75 min. Receptors were labeled with SNAP-Surface Alexa Fluor 649 at 4°C following each experimental condition and timepoint. Data are displayed as percentages of CCR2 detected relative to initial levels of surface CCR2 prior to stimulation (Start). (H, I) HEK293 cells expressing FLAG-CCR2 or FLAG-PAR1 were pretreated with 10 µg/mL cycloheximide for 90 min and left unstimulated (0 min) or stimulated with 100 nM CCL2 (to activate CCR2) or 100 μM TFLLRN (to activate PAR1) for 2 h at 37°C. Equivalent amount of cell lysates were immunoprecipitated with M2 anti-FLAG antibody and immunoblotting detection with anti-FLAG antibody. Cell lysates were analyzed for endogenous α-tubulin as controls (H). Receptor degradation was quantified and data (mean ± SEM) shown are expressed as the fraction of receptor remaining compared with untreated control cells as determined from three independent experiments (I). Data are expressed as the means ± SEM of n ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to controls by one-way analysis of variance (ANOVA) with Dunnet’s multiple comparison test or unpaired t test.

    Article Snippet: Subsequently, cells were washed and remaining non-internalized SNAP-CCR2 at the cell surface was blocked at 4°C with SNAP-Surface block (New England Biolabs) for 45 min.

    Techniques: Transfection, Stable Transfection, Expressing, Blocking Assay, Labeling, Immunoprecipitation, Western Blot

    Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).

    Journal: Molecular Metabolism

    Article Title: Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion

    doi: 10.1016/j.molmet.2022.101541

    Figure Lengend Snippet: Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).

    Article Snippet: SNAP substrates were purchased from NEB (SNAP-Surface® Block, S9143S; SNAP-Surface® Alexa Fluor® 546, S9132S; SNAP-Surface® Alexa Fluor® 647, S9136S).

    Techniques: Construct, Clone Assay, Western Blot, Incubation, Flow Cytometry, Labeling