Journal: Nature Neuroscience

Article Title: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

doi: 10.1038/s41593-024-01615-5

Figure Lengend Snippet: a , Immunofluorescence (IF) images of primary cortical neurons immunostained for glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2) simultaneously with PSD95 to show that the cultures are devoid of glial cells or oligodendrocytes, respectively. DAPI for nuclei; PSD95 for excitatory neurons. Magnification, ×40. Scale bars, 50 μm. b , TurboID-PSD95 was cloned without (top row) and with (bottom row) its 5′ and 3′ UTRs and lentivirally expressed in primary cortical neurons. White dashed boxes are zoomed in areas in black&white images. DAPI for nuclei; MAP2 for dendrites; Flag for each TurboID. % dendritically localized TurboID-PSD95 is quantified by co-localization with MAP2 signal in ImageJ. 3 different areas of images per replicate ( n = 3). Magnification, ×20. Scale bars, 50 μm. Significance was derived from biological replicates, showing the center line at mean. c , IF images of TurboID-PSD95-transduced neurons immunostained for DAPI (blue, for nuclei), PSD95 (red, for endogenous PSD95) and TurboID-PSD95 (cyan, detected by Flag). Magnification, ×60. Scale bar, 50 μm. d , IF images show the expression of a presynaptic marker, Synaptophysin (cyan), and TurboID-PSD95 (red, detected by Flag antibody) in primary cortical neurons transduced with TurboID-PSD95. DAPI (blue) marker for nuclei. Three zoomed in regions are marked by the white boxes. Magnification, ×60. Scale bar, 10 μm. e , IF images show TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 or Pan-TurboID after 30 minutes of biotin incubation. DAPI (blue, nuclei); MAP2 (green, dendrites); Flag (red, TurboID); and Streptavidin (cyan, biotinylated proteins). Magnification, ×20. Scale bars, 50 μm. f , Western blots stained for Flag and β-Actin from Pan-TurboID and TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin shown to indicate the relative expression levels of TurboID proteins. Quantifications of TurboID protein levels normalized to β-Actin are shown on the right ( n = 3); relative levels are not significant by two-tailed, paired Student’s t -test. g , Western blots stained for streptavidin signal in inputs (‘in’) and streptavidin pulldowns (‘pd’) from Pan-TurboID or TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. h , Streptavidin pulldowns shown for dendritic (SHANK3, GKAP, NLGN1 and HOMER1) and negative control (GAPDH) proteins from TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. Flag signal indicates self-biotinylation of each construct. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by TurboID-PSD95 in each condition is calculated by dividing the signal in the pulldown lane by that of the input lane, after each is adjusted to total, and quantifications are shown as bar graphs ( n = 3). P values: Flag = 0.58, SHANK3 = 0.0061, GKAP = 0.018, NLGN1 = 0.00052, HOMER1 = 0.021, GAPDH = 0.42. i , Streptavidin pulldowns shown for dendritic (BAIAP2 and DLGAP3) and nuclear (TBR1, H4 and H2AX) proteins from Pan-TurboID and TurboID-PSD95-transduced neurons in the presence (+) of exogenous biotin. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by each TurboID is calculated as in (h) ( n = 3). P values: BAIAP2 = 0.0052, DLGAP3 = 0.0035, TBR1 = 0.0063, H4 = 0.018, H2AX = 0.0037. j , Phosphorylation of EEF2, eIF2α, ERK1/2 and IRE1 and total levels of ATF4 and CHOP are shown in resting (rest), activated (DHPG, Dep) and stressed (Sodium arsenite (NaAsO 2 )) cells by using phospho-specific and total antibodies. The amount of phosphorylated or total protein is shown in the bar graphs, calculated by dividing the phosphorylated signal to total and β-Actin for the phosphorylated proteins and by dividing the total to β-Actin for ATF4 and CHOP ( n = 3). Significance was calculated with respect to rest. P values: P-EEF2 (DHPG = 0.0088, Dep = 0.0023, NaAsO 2 = 0.039), P-eIF2α (DHPG = 0.018, Dep = 0.0034, NaAsO 2 = 0.028), P-ERK1/2 (DHPG = 0.015, Dep = 0.0067, NaAsO 2 = 0.00084), P-IRE1 (DHPG = 0.06, Dep = 0.37, NaAsO 2 = 0.0027), ATF4 (DHPG = 0.038, Dep = 0.42, NaAsO 2 = 0.016), CHOP (DHPG = 0.044, Dep = 0.18, NaAsO 2 = 0.024). k , Quantitative PCR (qPCR) results shown for immediate early genes, Arc , Fos and Jun . The fold changes for each gene are calculated by first normalizing to the house-keeping gene β-Actin in each condition, then dividing the value of each condition by that of the resting state ( n = 3). l , Dendritic spine size in resting and KCl-depolarized neurons are measured using the Keyence microscope. Red squares are examples of spines that are counted ( n = 3, 12 spines from each biological replicate are counted as technical replicates). Significance was derived from the biological replicates using the two-tailed, unpaired Student’s t -test. Box plots show the min and max, with the center line at median. Magnification, ×100. Scale bars, 5 μm. m , Fluo-4-AM staining in resting, KCl-depolarized and DHPG-depolarized cells. Fluo4-AM was loaded in resting cells and measurements were taken at indicated time points after Fluo4-AM removal. In depolarized cells, the dye was loaded during silencing. After silencing, fluorescence was measured during stimulus at 10, 30 and 60-minute time points for the KCl treatment and at 10-minute for the DHPG-induced activation. Fluorescence was also measured 60 minutes after the stimulus removal (60′post KCl and 60′post DHPG). Circles represent data from 2 biological and 3 technical replicates. Below: Examples of Fluo4-AM fluorescence are shown in resting, 10-minute KCl-treated and 10-minute DHPG-treated neurons. Fluo4-AM loading (45 minutes) was performed during the last 45 minutes of the silencing step prior to stimulus addition for the KCl and DHPG treatment and simultaneously for the resting neurons. Imaging was performed 10 minutes after the stimulus was added. Scale bars, 50 μm. (b,f,h-k,m) Data are mean ± s.d. Significance was calculated using the two-tailed, paired Student’s t -test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Article Snippet: Puromycin (1:3,000, mouse, Kerafast, EQ0001, RRID: AB_2620162), Flag (1:3,000, mouse, Sigma-Aldrich, F1804, RRID: AB_262044), β-Actin antibody (1:2,500, mouse, Sigma-Aldrich, A1978, RRID: AB_476692), RPL10A (1:1,000, rabbit, Abcam, ab174318), MAP2 (1:2,500, guinea pig, Synaptic Systems, 188004, RRID: AB_2138181), GFAP (1:500, rabbit, Abcam, ab7260, RRID: AB_305808), OLIG2 (1:500, rabbit, Proteintech, 13999-1-AP, RRID: AB_2157541), PSD95 (1:500, mouse, Millipore, MABN68, RRID: AB_10807979), Synaptophysin (1:300, mouse, Abcam, ab8049, RRID: AB_2198854), SHANK3 (1:500, mouse, Novus, NBP1-47610, RRID: AB_10010567), GKAP (1:500, rabbit, Novus, NBP1-76911, RRID: AB_11017331), NLGN1 (1:200, mouse, Novus, NBP2-42192), HOMER1 (1:1,000, rabbit, Proteintech, 12433-1-AP, RRID: AB_2295573), GAPDH (1:5,000, mouse, Thermo Fisher Scientific, AM4300, RRID: AB_2536381), BAIAP2 (1:500, rabbit, Proteintech, 11087-2-AP, RRID: AB_2063075), DLGAP3 (1:500, rabbit, Proteintech, 55056-1-AP, RRID: AB_10858793), TBR1 (1:500, rabbit, Proteintech, 20932-1-AP, RRID: AB_10695502), H4 (1:1,000, mouse, Abcam, ab31830, RRID: AB_1209246), H2A.X (1:1,000, rabbit, Proteintech, 10856-1-AP, RRID: AB_2114985), EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2332, RRID:AB_10693546), P-EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2331, RRID: AB_10015204), eIF2α (1:1,000, rabbit, Cell Signaling Technology, 9722, RRID: AB_2230924), P-eIF2α (1:1,000, rabbit, Cell Signaling Technology, 3398, RRID: AB_2096481), p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 4695, RRID: AB_390779), P-p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 9101, RRID: AB_331646), P-IRE1 (1:500, rabbit, Novus, NB100-2323SS, RRID: AB_10145203), IRE1 (1:500, rabbit, Novus, NB100-2324SS, RRID: AB_10000972), CHOP (1:1,000, mouse, Cell Signaling, 2895T, RRID: AB_2089254), ATF4 (1:1,000, rabbit, Cell Signaling Technology, 11815S, RRID: AB_2616025), MPHOSPH (1:300, rabbit, Biorbyt, orb100446), KCNJ9 (1:300, rabbit, LSBio, LS-C352416), KCNJ9 (1:300, mouse, Antibodies Incorporated, 75-445, RRID: AB_2686912), eIF4G2 (1:1,000, rabbit, Cell Signaling Technology, RRID: AB_10622189 and rabbit, Cell Signaling Technology, RRID: AB_2261993), NSUN3 (1:250, rabbit, LSBio, LS-C163024), MTF1 (1:300, rabbit, Novus, NBP1-86380, RRID: AB_11011361), ZFP64 (1:300, rabbit, Proteintech, 17187-1-AP, RRID: AB_2218826) and KATNBL1 (1:250, rabbit, Proteintech, 24795-1-AP, RRID: AB_2879730).

Techniques: Immunofluorescence, Clone Assay, Derivative Assay, Expressing, Marker, Transduction, Incubation, Western Blot, Staining, Two Tailed Test, Negative Control, Construct, Isolation, Real-time Polymerase Chain Reaction, Microscopy, Fluorescence, Activation Assay, Imaging

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Relative expression of Gpr31b in tissues from C57BL/6J mice by quantitative polymerase reaction (PCR) (n=3 mice per condition). BMDM: bone-marrow derived macrophages; pMacs: peritoneal macrophages. (B) Schematic of approach to whole-body KO of Gpr31b . (C) Representative genotypes of wild type (WT) and Gpr31b KO mice by PCR. The WT Gpr31b allele generates an amplicon of 335 bp and the mutant allele generates an amplicon of 699 bp. Additionally, there is a WT band that includes pseudogenes with an amplicon of 3100 bp. (D) Relative expression of Gpr31b in WT, heterozygous, and Gpr31b KO mice by quantitative PCR (n=3 mice per condition). (E) Sequencing of PCR products of amplification of Gpr31b and its pseudogenes (n=3 mice per condition). (F) Body weight of 8-week-old males. (G) Glucose tolerance testing (GTT) of 8-week-old males ( left panel ) with area under the curve (AUC, right panel ). (H) GTT of 8-week-old males ( left panel ) with AUC (right panel). (I) Body weight of 8-week-old females. (J) GTT of 8-week-old females ( left panel ) with AUC ( right panel ). (K) GTT of 15-week-old females ( left panel ) with AUC ( right panel ). Data presented as mean with SEM. Single data points represent individual mice. Statistical significance determined by Student’s t-test or one-way analysis of variance (ANOVA).

Article Snippet: Quantitative PCR was performed using Bio-Rad CFX Opus with a predesigned Taqman assay probe for mouse genes Gpr31 (Mm02391728_g1) and Actb (Mm01205647_m1) (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Derivative Assay, Amplification, Mutagenesis, Real-time Polymerase Chain Reaction, Sequencing

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Schematic of experimental approach for RNA sequencing of islets treated with vehicle or pro-inflammatory cytokines (PIC). (B) RNA sequencing data showing relative expression of Gpr31b in WT and KO islets treated with PIC (n=3 mice per condition). (C) Principal components analysis (PCA) of WT and Gpr31b KO islets treated with vehicle (n=3 mice per condition). (D) Volcano plot of differentially expressed genes in WT and Gpr31b KO islets treated with vehicle. Genes which meet the threshold of absolute fold change ≥1.5 and adjusted P-value <0.05 are in red; genes which only meet the adjusted P-value threshold are in blue. (E) Principal components analysis (PCA) of WT and Gpr31b KO islets treated with PIC (n=3 mice per condition). (F) Volcano plot of differentially expressed genes in WT and Gpr31b KO islets treated with PIC (n=3 mice per condition). Genes which meet the threshold of absolute fold change ≥1.5 and adjusted P-value <0.05 are in red; genes which only meet the adjusted P-value threshold are in blue. (G) Analysis of CD5L concentration by enzyme-linked immunosorbent assay (ELISA) in the supernatant of islets treated with vehicle or PIC. (H) Gene set enrichment analysis (GSEA) of WT and Gpr31b KO islets treated with PIC (n=3 mice per condition) using hallmark gene sets. Both suppressed ( top panel ) and activated gene sets ( bottom panel ) are shown. Normalized enrichment score (bars) and log-transformed P-value (blue dots). All pathways meeting significance criteria of adjusted P-value <0.05 are shown. (I) Heatmap from WT and Gpr31b KO islets treated with PIC showing Z-scores for differentially expressed genes meeting more stringent criteria of absolute fold change ≥2 with adjusted P-value <0.05. (J) Heatmap from WT and Gpr31b KO islets treated with PIC showing Z-scores for genes in the leading edge for the ROS hallmark gene set. Data presented as mean with SEM. Statistical significance determined by Student’s t-test or paired one-way analysis of variance (Friedman ANOVA).

Article Snippet: Quantitative PCR was performed using Bio-Rad CFX Opus with a predesigned Taqman assay probe for mouse genes Gpr31 (Mm02391728_g1) and Actb (Mm01205647_m1) (Invitrogen, Carlsbad, CA).

Techniques: RNA Sequencing, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Gene set enrichment analysis (GSEA) of WT and Gpr31b KO islets treated with vehicle (n=3 mice per condition) using hallmark gene sets. Both suppressed ( top panel ) and activated gene sets ( bottom panel ) are shown. Normalized enrichment score (bars) and log-transformed P-value (blue dots). All pathways meeting significance criteria of adjusted P-value <0.05 are shown. (B) Suppressed gene sets from GSEA of WT and Gpr31b KO islets treated with cytokines using gene ontology (GO) annotations. (C) Activated gene sets from GSEA of WT and Gpr31b KO islets treated with cytokines using GO annotations. Normalized enrichment score (bars) and log-transformed P-value (blue dots).

Article Snippet: Quantitative PCR was performed using Bio-Rad CFX Opus with a predesigned Taqman assay probe for mouse genes Gpr31 (Mm02391728_g1) and Actb (Mm01205647_m1) (Invitrogen, Carlsbad, CA).

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Representative fluorescence images of WT and Gpr31b KO islets treated with vehicle or PIC. Imaging shows nuclei stained by 4’,6-diamidino-2-phenylindole (DAPI; blue) and CellROX (magenta). Scale bars are 100 µm. (B) Quantification of CellROX mean intensity for the experiment in (A) (n=5-7 mice). Data presented as mean with SEM. Single data points represent individual mice. Statistical significance determined by one-way ANOVA.

Article Snippet: Quantitative PCR was performed using Bio-Rad CFX Opus with a predesigned Taqman assay probe for mouse genes Gpr31 (Mm02391728_g1) and Actb (Mm01205647_m1) (Invitrogen, Carlsbad, CA).

Techniques: Fluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Schematic of experimental approach for polarization of macrophages. (B) Flow cytometry for the M1 marker iNOS as a percentage of F4/80+ cells in macrophages from WT and Gpr31b KO mice (n=5-6 mice per condition). (C) Flow cytometry for the M2 marker CD206 as a percentage of F4/80+ cells in macrophages from WT and Gpr31b KO mice (n=6 mice per condition). (D) Relative expression of Gpr31b from both M0 and M1-like macrophage by RNA sequencing (n=3 mice per condition). (E) Volcano plot of differentially expressed genes in M0 macrophages from WT and Gpr31b KO mice (n=3 mice per condition). Genes which meet the threshold of absolute fold change ≥1.5 and adjusted P-value <0.05 are in red; genes which only meet the adjusted P-value threshold are in blue. (F) Volcano plot of differentially expressed genes in M1-like macrophages from WT and Gpr31b KO mice (n=3 mice per condition). Genes which meet the threshold of absolute fold change ≥1.5 and adjusted P-value <0.05 are in red; genes which only meet the adjusted P-value threshold are in blue. (G) GSEA of M1-like macrophages from WT and Gpr31b KO mice (n=3 mice per condition) using hallmark gene sets. Both suppressed ( top panel ) and activated gene sets ( bottom panel ) are shown. Normalized enrichment score (bars) and log-transformed P-value (blue dots). All significant pathways are shown. (H) Leading edge genes in M1-like macrophages from WT and Gpr31b KO mice from the IFN-γ and the IFN-α response hallmark gene sets. Genes represented in both leading edges are in the far left heatmap. (I) Schematic of BMDM transwell migration assay. (J) Quantification of transwell migration assay of BMDMs from WT and Gpr31b KO mice (n=3 mice per condition). (K) Schematic of zebrafish tailfin cut assay. (L) Representative images of zebrafish tailfin cut assay showing DNA (TO-PRO; yellow) and macrophages (GFP; green) ( left panel ). Scale bars are 100 µm. Quantification of assay results as macrophage count ( right panel ) (n=26-32 zebrafish per condition). Data presented as mean with SEM. Statistical significance determined by Student’s t-test or one-way ANOVA.

Article Snippet: Quantitative PCR was performed using Bio-Rad CFX Opus with a predesigned Taqman assay probe for mouse genes Gpr31 (Mm02391728_g1) and Actb (Mm01205647_m1) (Invitrogen, Carlsbad, CA).

Techniques: Flow Cytometry, Marker, Expressing, RNA Sequencing, Transformation Assay, Transwell Migration Assay

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Schematic of multiple low-dose STZ (mldSTZ)-induced diabetes experiment. (B) Blood glucose measurements for WT (black dots) and Gpr31b KO (blue dots) mice for 30 days after STZ administration (n=5-6 mice per condition). GTT at 10 days after STZ is also indicated (C) GTT at 10 days after STZ administration ( left panel ) with AUC ( right panel ). (D) Representative immunohistochemical labeling of insulin in pancreatic tissue from WT and Gpr31b KO mice ( left panel ). Scale bars are 250 µm. Quantification of β-cell mass ( right panel ) (n=5 mice per condition). (E) Representative immunofluorescence labeling for nuclei (DAPI; blue), insulin (teal), and the oxidative stress marker 4-HNE (magenta) ( left panel ). Scale bars are 50 µm. Quantification of 4-HNE mean intensity (n=17-51 islets per condition) ( right panel ). (F) Immunohistochemical labeling for the macrophage marker F4/80 in pancreatic tissue from WT and Gpr31b KO mice ( left panel ). Scale bars are 200 µm. Quantification of infiltrated (intra-islet) macrophages per area (n=14-28 islets per condition) ( right panel ). Islets are marked with a dotted line and infiltrated (intra-islet) macrophages are marked with arrowheads. Data presented as mean with SEM. Statistical significance determined by one-way ANOVA.

Article Snippet: Quantitative PCR was performed using Bio-Rad CFX Opus with a predesigned Taqman assay probe for mouse genes Gpr31 (Mm02391728_g1) and Actb (Mm01205647_m1) (Invitrogen, Carlsbad, CA).

Techniques: Immunohistochemical staining, Labeling, Immunofluorescence, Marker

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Relative Gpr31b mRNA expression in MIN6 murine β cells treated with vehicle or PIC as well as non-targeting control pool siRNA (siNTC or NTC) and siRNA against Gpr31b (si Gpr31b ). (B) Schematic of siRNA injection experiment in NOD mice. (C) Representative images of immunohistochemistry for insulin in pancreatic tissue from NOD mice injected with siNTC or si Gpr31b ( top left panel ). Scale bars are 250 µm. Representative immunofluorescence labeling from the same tissues for nuclei (DAPI; blue), insulin (green), and glucagon (magenta) ( bottom left panel ). Scale bars are 100 µm. Quantification of β-cell mass ( right panel ) (n=5 mice per condition). (D) Mean insulitis score for NOD mice injected with siNTC or si Gpr31b (n=5 mice per condition). (E) Representative immunofluorescence labeling in pancreatic tissue from siNTC or si Gpr31b injected mice showing nuclei (DAPI; blue), insulin (green), and the oxidative marker 4-HNE (magenta) ( left panel ). Scale bars are 100 µm; islets are marked with a dotted line. Quantification of 4-HNE mean intensity (n=5 mice per condition) ( right panel ). (F) Representative immunohistochemistry for the macrophage marker F4/80 in pancreatic tissue from NOD mice injected with siNTC or si Gpr31b (n=38-42 islets per condition) ( left panel ). Islets are marked with a dotted line, and representative infiltrated (intra-islet) macrophages are marked with arrowheads. Scale bars are 100 µm. Quantification of number of F4/80+ macrophages within the islet area ( right panel ). Data presented as mean with SEM. Statistical significance determined by Student’s t-test.

Article Snippet: Quantitative PCR was performed using Bio-Rad CFX Opus with a predesigned Taqman assay probe for mouse genes Gpr31 (Mm02391728_g1) and Actb (Mm01205647_m1) (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Control, Injection, Immunohistochemistry, Immunofluorescence, Labeling, Marker

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Flow cytometry from splenic tissue of NOD mice injected with siNTC or si Gpr31b . (B) Flow cytometry from pancreatic lymph nodes (pLN) of NOD mice injected with siNTC or si Gpr31b .

Article Snippet: Quantitative PCR was performed using Bio-Rad CFX Opus with a predesigned Taqman assay probe for mouse genes Gpr31 (Mm02391728_g1) and Actb (Mm01205647_m1) (Invitrogen, Carlsbad, CA).

Techniques: Flow Cytometry, Injection