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. 2022 Jun 20;24(7):1038–1048. doi: 10.1038/s41556-022-00931-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 7 — a. Normalized ratio of baseline calcium level measured by Indo-1 followed by calcium flux in response to D-glucuronic acid (GA), glucosamine hydrochloride (G), and neuromedin B (NMB) in GPRC5C-OE (n = 3 experiments) and control (n = 4 experiments) in AML3 cells. b. HA and GPRC5C expression in AML3 and AML5. c. HAS1,2, and 3 expression in AML3 and AML5. n = 3 experiments, except for HAS3 expression in AML3 n = 2 experiments. d. Technical PLA controls of GPRC5C and HA in AML3 and AML5. Plot is representative of one out of three independent experiments. Each independent experiment was performed in two technical replicates. n = 543, 551, 463, 990, 503, and 523 cells for AML3: GPR + HA, GPR only, HA only and AML5: GPR + HA, GPR only, and HA only, respectively. Data presented as mean ± SEM. e. Concentration–response curve of PRESTO-Tango construct of GPRC5C and control vector treated with GA, G, and NMB in HTLA cells. Normalized to luciferase response at the lowest concentration. n = 4 experiments. f. Residue conservation of GPRC5C based on a multiple sequence alignment of 250 homologous proteins extracted from UniRef90. Red: highly conserved; purple: conserved; blue: not conserved; light blue: no conservation information available; yellow background: sequon for N-glycosylation used for mutation analysis within the ECD of the protein; black box: the putative BX₇B domain. g. FACS validation of surface protein levels of GPRC5C^WT and site-specific mutant (GPRC5C^131QQ132) in HTLA cells. h. FACS validation of surface protein levels of GPRC5C^WT and site-specific mutant (GPRC5C^131AA132) in HTLA cells. i. Concentration-response curve of PRESTO-Tango construct for GPRC5C^WT and site-specific mutant (GPRC5C^131AA132) at predicted HA interaction site in HTLA cells. n = 6 experiments. j. FACS validation of surface protein levels of GPRC5C^WT and site-specific mutants (GPRC5C^N53L and GPRC5C^N203L) in HTLA cells. k. HA-fluorescein-treated cells quantified for GFP retention in HEK293T cells expressing GPRC5C^WT and GPRC5C^N53L. FACS plots representing HA retention in each condition. n = 4 experiments, except for GPRC5C^WT where n = 7 experiments. l. Concentration-response curve of PRESTO-Tango construct for GPRC5C^WT and site-specific mutant (GPRC5C^N53L) at predicted HA interaction site in HTLA cells. n = 6 experiments. m. Concentration-response curve of PRESTO-Tango construct for GPRC5C^WT treated with tunicamycin in HTLA cells. n = 3 experiments. n. Concentration-response curve of PRESTO-Tango construct for GPRC5C^WT treated with PNGase in HTLA cells. n = 3 experiments. All data presented as mean ± SD, except where otherwise stated. Statistical significance was determined using two-tailed t-test. ns, not significant. n indicates the number of replicates. For all experiments, at least two independent experiments were performed. Source numerical data are available in source data.

Source data