Journal: Cancer Medicine

Article Title: Exploring the Relationship Between Adipocytokines and Endometrial Cancer: Identifying Correlations With Clinico‐Pathological Prognostic Factors

doi: 10.1002/cam4.71007

Figure Lengend Snippet: (A, B) Bar‐graph demonstrating the expression of (A) biomarkers and (B) their receptors in endometrial cancer tissue ( n = 39). X axis‐ biomarkers (A), receptors (B). Y axis–expressions of biomarkers as fold change over benign endometrial calibrator sample (assigned value = 1, for comparison with the cancer samples). The error bars represent standard errors of mean. 1 (C) Heat map demonstrating the expression of all 6 biomarkers and their receptors in endometrial cancer tissues ( n = 39). AdipoQ, adiponectin; ADIPOR1/R2, adiponectin receptors; OBR, leptin receptor; TNF1A, TNFRSF1A; TNF1B, TNFRSF1B (TNF receptors). Gene expressions are plotted on the Y ‐axis against the individual patients on the X ‐axis. Gene expressions are plotted as fold changes over a single pooled calibrator sample value. The color scale ranges from black (lowest value) to white (median) to yellow (highest value).

Article Snippet: The antibodies used were: ADIPOR1 (Proteintech, Manchester, UK, #66619‐1‐Ig; anti‐mouse antibody) and ADIPOR2 (Abcam, Cambridge, UK, #ab223752; anti‐rabbit antibody).

Techniques: Expressing, Comparison

Journal: Cancer Medicine

Article Title: Exploring the Relationship Between Adipocytokines and Endometrial Cancer: Identifying Correlations With Clinico‐Pathological Prognostic Factors

doi: 10.1002/cam4.71007

Figure Lengend Snippet: Heat map comparing the expression of (A) adiponectin, leptin and their receptors in endometrial cancer tissue and parametrial adipose tissue ( n = 39) (B) all 6 biomarkers and their receptors in endometrial cancer tissue and lymph nodes ( n = 12). AdipoQ, adiponectin; AdipoR1/R2, adiponectin receptors; ObR, leptin receptor; TNF1A, TNFRSF1A; TNF1B, TNFRSF1B (TNF receptors). Gene expressions are plotted on the Y ‐axis against the individual patients on the X ‐axis. Gene expression plotted as fold changes over a single pooled calibrator sample. The color scale ranges from black (lowest value) to white (median) to yellow (highest value).

Article Snippet: The antibodies used were: ADIPOR1 (Proteintech, Manchester, UK, #66619‐1‐Ig; anti‐mouse antibody) and ADIPOR2 (Abcam, Cambridge, UK, #ab223752; anti‐rabbit antibody).

Techniques: Expressing, Gene Expression

Journal: Cancer Medicine

Article Title: Exploring the Relationship Between Adipocytokines and Endometrial Cancer: Identifying Correlations With Clinico‐Pathological Prognostic Factors

doi: 10.1002/cam4.71007

Figure Lengend Snippet: Illustration of chromogenic 3,3′‐Diaminobenzidine (DAB) staining for ADIPOR1 (A) and ADIPOR2 (B) receptor in endometrial cancer tissue and benign tissues. Image taken at 10× magnification, Scalebar = 100 μm.

Article Snippet: The antibodies used were: ADIPOR1 (Proteintech, Manchester, UK, #66619‐1‐Ig; anti‐mouse antibody) and ADIPOR2 (Abcam, Cambridge, UK, #ab223752; anti‐rabbit antibody).

Techniques: Staining

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 2 AdipoR1 mediates the adiponectin-induced increase in IT. A, B Protein abundance of adipoR1 (A) and adipoR2 (B) in the brain and TGs of intact mice. GAPDH was used as an equal loading control. Representative blots of at least 3 independent experiments are shown. C, Colocalization of adipoR1 (red) with NeuN, GS, NF200, CGRP, IB4 or Cav3.2 (green) in TG sections. Arrows show the colocalization. Scale bar, 50 µm. D, Protein abundance of adipoR1 in TG cells treated with control siRNA (NC-siRNA) or adipoR1 siRNA (adipoR1-siRNA). Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. NC-siRNA), unpaired t test. E, Representative traces (left) and bar chart (right) revealing that adipoR1-siRNA treatment prevented the adiponectin-induced IT increase (n = 11 cells). Adiponectin at 100 nM significantly enhanced IT in cells transduced with NC-siRNA (n = 12 cells). **p < 0.01 (vs. control + NC-siRNA), unpaired t test

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Quantitative Proteomics, Control, Transduction

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 3 The AdipoR1-induced IT increase is mediated by the protein kinase CK2α. A Time course of IT changes (left) and bar chart (right) showing the effect of adiponectin at 100 nM on IT in cells dialyzed with GDP-β-S (1 mM) (n = 11 cells). Insets indicate representative current traces. Numbers represent points used for exemplary traces. B Coimmunoprecipitation of adipoR1 with RACK1 in the TG. Representative blots of at least 3 independent experiments are shown. C Protein abundance of RACK1 in TG cells treated with RACK1-siRNA or NC-siRNA. Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. NC-siRNA), unpaired t test. D Summary of results revealing that treatment with either NC-siRNA (n = 10 cells) or RACK1-siRNA (n = 11 cells) did not affect the adiponectin-induced IT response. *p < 0.05 (vs. control + NC-siRNA), #p < 0.05 (vs. control + RACK1-siRNA), unpaired t test. E Interaction of adipoR1 with PKCK2α in the TG. Representative blots of at least 3 independent experiments are shown. F-G Time course of IT changes showing the effect of adiponectin at 100 nM on IT in cells preincubated with CX-4945 (F, 10 μM) or TBB (G, 20 μM). Insets indicate representative current traces. Numbers represent points used for exemplary traces. H Summary of the results revealing the effect of adiponectin at 100 nM on IT in the presence of CX-4945 (n = 8 cells) or TBB (n = 11 cells). ***p < 0.001 (vs. control), paired t test. I Protein abundance of CK2α in NC-siRNA- or CK2α-siRNA-treated groups. Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. NC-siRNA), unpaired t test. J Representative traces (left) and bar chart (right) revealing that treatment with CK2α-siRNA prevented the adiponectin-induced IT increase (n = 11 cells). Adiponectin at 100 nM significantly enhanced IT in cells transduced with NC-siRNA (n = 12 cells). *p < 0.05 (vs. control + NC-siRNA), unpaired t test

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Quantitative Proteomics, Control, Transduction

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 5 PKCβ1 is involved in the adipoR1-induced IT response. A Determination of mRNAs of classic PKC isoforms (PKCα, PKCβ1, PKCβ2 and PKCγ) in the TG of mice. No signal was detected in the reactions without RT (− RT). B Bar graph revealing the effect of 100 nM adiponectin on IT in the presence of LY333531 (200 nM, n = 10 cells), HBDDE (2 µM, n = 8 cells), PKCβ1 inhibitory peptide (PKCβ1-IP, 10 µM, n = 9 cells), or PKCβ2-IP (10 µM, n = 9 cells). ***p < 0.001 (vs. control), paired t test. C Protein abundance of PKCβ1 in TG cells treated with NC-siRNA or PKCβ1-siRNA. Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. NC-siRNA), unpaired t test. D Bar graph revealing that treatment with PKCβ1-siRNA prevented the adiponectin-induced IT increase (n = 9 cells). Adiponectin at 100 nM still significantly enhanced IT in cells transduced with NC-siRNA (n = 12 cells). *p < 0.05 (vs. control + NC-siRNA), unpaired t test. E Immunofluorescence analysis of PKCβ1 translocation mediated by 100 nM adiponectin. Arrows in white indicate the line-scanned area. Data are representative of 3 independent experiments. F Immunoblot analysis of PKCβ1 expression in cytoplasmic and membrane fractions isolated from TG cells treated with 100 nM adiponectin. GAPDH served as a control for protein loading. α-Na+/K+ ATPase was used as an indicator for membrane contamination of cytosolic extracts. Representative blots of at least 3 independent experiments are shown. **p < 0.01 (vs. control), unpaired t test

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Control, Quantitative Proteomics, Transduction, Immunofluorescence, Translocation Assay, Western Blot, Expressing, Membrane, Isolation

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 8 Peripheral adipoR1 participated in mechanical pain hypersensitivity. A Escape threshold after intra-TG injection of vehicle or adiponectin at 0.1 nmol, 1 nmol or 5 nmol. *p < 0.05 (vs. vehicle) at the corresponding time point, two-way ANOVA. B, C Administration of adipoR1-siRNA (B) or PKCβ1-siRNA (C) prevented adiponectin-induced mechanical hypersensitivity. *p < 0.05 (vs. vehicle); #p < 0.05 (vs. APN) in NC-siRNA-treated groups, two-way ANOVA. D Pretreatment with TTA-P2 (1 nmol) or Z941 (0.5 nmol) attenuated adiponectin (1 nmol)-induced mechanical hypersensitivity. *p < 0.05 (vs. vehicle), #p < 0.05 (vs. APN) at the 1-h time point, two-way ANOVA. E Escape threshold to mechanical stimuli in the sham- or CCI-ION-operated groups. ***p < 0.01 (vs. sham) at the corresponding time point, two-way ANOVA. F Protein abundance of adipoR1 in TGs 14 days following CCI-ION or sham surgery. *p < 0.05 (vs. sham), unpaired t test. Representative blots of at least 3 independent experiments are shown. G Intra-TG administration of adipoR1-siRNA 14 days after CCI-ION significantly attenuated mechanical hypersensitivity in CCI-ION mice. *p < 0.05 and **p < 0.01 (vs. NC-siRNA), two-way ANOVA. H Effect of Cav3.2-siRNA vs. NC-siRNA (Day 0) on adipoR1-siRNA (intra-TG injection on Day 3)-induced alleviation of mechanical allodynia in CCI-ION mice. Intra-TG injection of adipoR1-siRNA did not have additive effects to Cav3.2-siRNA on mechanical allodynia in CCI-ION mice. ** p < 0.01 (vs. CCI-ION) at -14 days, # p < 0.05 and ## p < 0.01 (vs. NC-siRNA) at the 3-day point in CCI-ION mice, + p < 0.05 (vs. Cav3.2-siRNA) at the 0-day point in CCI-ION mice, two-way ANOVA

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Injection, Quantitative Proteomics

Journal: The journal of headache and pain

Article Title: Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

doi: 10.1186/s10194-023-01658-2

Figure Lengend Snippet: Fig. 9 Illustration of proposed mechanisms of adipoR1 signaling on Cav3.2 channels. Adiponectin acts through the G protein-independent adipoR1, leading to the activation of protein kinase CK2α subunits. CK2α stimulates the downstream conventional PKCβ1, which selectively modulates Cav3.2 channel activity, resulting in an IT increase. The signaling cascade mediated by adipoR1 contributes to TG neuronal hyperexcitability and nociceptive behaviors of adiponectin. Importantly, adipoR1 was upregulated in the injured TG, and blockade of Cav3.2 attenuated adipoR1-mediated pain hypersensitivity in CCI-ION-induced neuropathic pain behaviors. Neither PKA, CaMKII nor novel PKC isoforms are involved in the adiponectin-mediated IT response. Nevertheless, whether PKCβ1 directly phosphorylates Cav3.2 channels or acts via some intermediate signaling molecules needs to be further explored

Article Snippet: The human adipoR1 cDNA obtained from Origene was cloned into the pCMV6-AC-GFP vector.

Techniques: Activation Assay, Activity Assay

Journal: Scientific Reports

Article Title: ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

doi: 10.1038/s41598-018-32579-9

Figure Lengend Snippet: ADIPOR1 protein expression in mouse tissues. ( a ) Anti-ADIPOR1 antibody (IBL) can recognize endogenous and exogenous ADIPOR1. Lanes: 1 – Flag-ADIPOR1 transfected HEK293T, 2 – WT/Untransfected HEK293T, 3 – CRISPR negative control transfected HEK293T, 4 – ADIPOR1 KO HEK293T clone 4, ADIPOR1 KO HEK293T clone 5. Anti-Flag antibody (CST) was used to detect exogenous tagged ADIPOR1. CYCLOPHILIN B was used as a loading control. ( b ) ADIPOR1 protein expression profile from different tissues. Adult mouse tissues and P22 mouse eye tissue were profiled. ADIPOR1 is enriched in the eye and brain. The antibody retains specificity in tissue samples as based on WT and KO eye tissue discrimination. ( c ) ADIPOR1 protein is present in non-nervous tissues. ADIPOR1 protein signal can be appreciated in liver, heart, and skeletal muscle when ran without brain or eye samples, confirming protein presence in those tissues. Four and a half month old AdipoR1 WT and KO mice were used for tissue collection. Each lane represents a sample from an individual mouse. VINCULIN was used as a loading control. For ( a ) and ( c ) each membrane was cut and probed separately for the analyzed protein and the loading control.

Article Snippet: To generate exogenous ADIPOR1 expressing cells, HEK293T cells were plated as 3 million cells per 10 cm plate the day before transfection, and transfected using 0.5 µg of Flag-ADIPOR1 expression plasmid (Cat # HG14109-NF, Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific).

Techniques: Expressing, Transfection, CRISPR, Negative Control

Journal: Scientific Reports

Article Title: ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

doi: 10.1038/s41598-018-32579-9

Figure Lengend Snippet: ADIPOR1 is expressed in the neural retina and RPE. IHC for ADIPOR1 protein expression. P21 mouse eyes of AdipoR1 WT or KO genotypes were stained with the anti-ADIPOR1 antibody (IBL). ADIPOR1 signal is observed in the neural retina and the RPE with strongest expression in the OS. Scale bar = 20 µm. Z-stacks were acquired and the maximum intensity projection is displayed.

Article Snippet: To generate exogenous ADIPOR1 expressing cells, HEK293T cells were plated as 3 million cells per 10 cm plate the day before transfection, and transfected using 0.5 µg of Flag-ADIPOR1 expression plasmid (Cat # HG14109-NF, Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific).

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

doi: 10.1038/s41598-018-32579-9

Figure Lengend Snippet: Three week old AdipoR1 KO mice exhibit deficient expression of visual system proteins. ( a ) Western blots of P22 AdipoR1 WT, HET, and KO mouse eyes are shown. ADIPOR1 as well as known visual system proteins were profiled. VINCULIN was used as a loading control. Each lane represents a sample from an individual mouse, n = 3. KO mice exhibit reduced levels of RHODOPSIN, PERIPHERIN-2, ROM1, PDE6α, and GNAT1. Each membrane was cut and probed separately for the analyzed protein and the loading control. ( b ) Densitometry quantification of western blots in (a). ANOVA (α = 0.05) with Dunnett’s multiple comparisons test (WT set as control) was used. Error bars represent standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS = not significant. P values: ADIPOR1, WT vs HET p = 0.0409, WT vs KO p = 0.0008; CRX = WT vs HET p = 0.0811, WT vs KO p = 0.3451; PDE6α, WT vs HET p = 0.0045, WT vs KO p = 0.0001; RHODOPSIN = WT vs HET p = 0.2541, WT vs KO p = 0.0058; GNAT1 = WT vs HET p = 0.0706, WT vs KO p = 0.0298; RPE65 = WT vs HET p = 0.4107, WT vs KO p = 0.2346; PERIPHERIN2 = WT vs HET p = 0.4984, WT vs KO p = 0.0473; ROM1 = WT vs HET p = 0.0825, WT vs KO p = 0.0409.

Article Snippet: To generate exogenous ADIPOR1 expressing cells, HEK293T cells were plated as 3 million cells per 10 cm plate the day before transfection, and transfected using 0.5 µg of Flag-ADIPOR1 expression plasmid (Cat # HG14109-NF, Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

doi: 10.1038/s41598-018-32579-9

Figure Lengend Snippet: Two week old AdipoR1 KO mice appear normal. ( a ) Western blots of P15 AdipoR1 WT, HET, and KO mouse eyes are shown. ADIPOR1 as well as known visual system proteins were profiled. VINCULIN was used as a loading control. Each lane represents a sample from an individual mouse, n = 7 for WT, n = 8 for HET, n = 8 for KO. KO mice appear normal except for a possible decrease of RPE65. Each membrane was cut and probed separately for the analyzed proteins and the loading control. ( b ) Densitometry quantification of western blots in (a). ANOVA (α = 0.05) with Dunnett’s multiple comparisons test (WT set as control) was used. Error bars represent standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS = not significant. P values: ADIPOR1, WT vs HET p = 0.0001, WT vs KO p = 0.0001; RPE65, WT vs HET p = 0.9917, WT vs KO p = 0.0261; RHODOPSIN, WT vs HET p = 0.8916, WT vs KO p = 0.8314; PERIPHERIN2, WT vs HET p = 0.3510, WT vs KO p = 0.3317; PDE6α, WT vs HET p = 0.3728, WT vs KO p = 0.7756; GNAT1, WT vs HET p = 0.5035, WT vs KO p = 0.9541.

Article Snippet: To generate exogenous ADIPOR1 expressing cells, HEK293T cells were plated as 3 million cells per 10 cm plate the day before transfection, and transfected using 0.5 µg of Flag-ADIPOR1 expression plasmid (Cat # HG14109-NF, Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific).

Techniques: Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

doi: 10.1038/s41598-018-32579-9

Figure Lengend Snippet: AAV- Cre treated adult floxed mice show decreased expression of markers critical for vision. ( a ) Western blot for RHODOPSIN of IRBP-Cre treated mouse eyes is shown. VINCULIN or α-TUBULIN was used as a loading control. Each lane represents an individual eye from a mouse of that genotype, n = 10 for WT, n = 14 for Floxed. Densitometry quantification for RHODOPSIN and additional western blots (see Supplementary Fig. ) is shown below. IRBP-Cre treated mice show a large drop in ADIPOR1, confirming Cre induced KO of this protein. These mice also display a significant drop of RHODOPSIN and RPE65. IRBP group - P values: ADIPOR1 - p = < 0.0001; RHODOPSIN - p = 0.0033; CRX - p = 0.0512; RPE65 - p = 0.0009; GNAT1 - p = 0.2680; IRBP - p = 0.0570. ( b ) Western blot for RHODOPSIN of VMD2-Cre treated mouse eyes is shown. VINCULIN or α-TUBULIN was used as a loading control. Each lane represents an individual eye from a mouse of that genotype, n = 10 for WT, n = 11 for Floxed. Densitometry quantification for RHODOPSIN and additional western blots (see Supplementary Fig. ) is shown below. VMD2-Cre treated mice show a small drop in ADIPOR1 consistent with a KO of this protein only in the RPE layer. These mice also display a significant drop of RHODOPSIN, CRX, GNAT1, and IRBP. VMD2 group - P values: ADIPOR1 - p = 0.0080; RHODOPSIN - p = 0.0092; CRX - p = 0.0093; RPE65 - p = 0.3471; GNAT1 - p = 0.0087; IRBP - p = 0.0442. Unpaired two tailed t-test was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS = not significant. Error bars represent standard deviation. Mice were taken down at 5 months post AAV injection. For western blots in ( a , b ) each membrane was cut and probed separately for the analyzed protein and the loading control.

Article Snippet: To generate exogenous ADIPOR1 expressing cells, HEK293T cells were plated as 3 million cells per 10 cm plate the day before transfection, and transfected using 0.5 µg of Flag-ADIPOR1 expression plasmid (Cat # HG14109-NF, Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific).

Techniques: Expressing, Western Blot, Two Tailed Test, Standard Deviation, Injection

Journal: Scientific Reports

Article Title: ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

doi: 10.1038/s41598-018-32579-9

Figure Lengend Snippet: Mfrp rd6 mice lack ADIPOR1 in their RPE layer. ( a ) IHC on P32 Mfrp wt and Mfrp rd6 mouse eyes. Anti-ADIPOR1 staining was performed on Mfrp wt controls and Mfrp rd6 mouse eyes. Anti-MFRP staining below confirms genotypes. Inset shows enlarged image near RPE layer. ADIPOR1 signal is lost in the RPE of Mfrp rd6 animals. ADIPOR1/MFRP is observed via the red reaction product. Scale bar = 20 µm. ( b ) Western blot for ADIPOR1 using Mfrp wt and Mfrp rd6 mouse eyes of age P21. α-TUBULIN was used as a loading control. Each lane represents an eye from a mouse of that genotype and eyes were loaded in pairs from individual animals (5 Mfrp wt and 5 Mfrp rd6 individual animals), n = 10 eyes for both genotypes. The membrane was cut and probed separately for the analyzed protein and the loading control. Below, a zoomed in image of lanes 8–13 highlights the absence of the top ADIPOR1 band from the Mfrp rd6 animals. ( c ) Western blot for ADIPOR1 comparing samples from a mouse eye, human stem cell derived RPE, and HEK293T cells. RPE and HEK293T ADIPOR1 runs at the same molecular weight as the top band from the mouse eye ADIPOR1 doublet. RPE cells were derived from H9 hESCs and matured for three months before analysis.

Article Snippet: To generate exogenous ADIPOR1 expressing cells, HEK293T cells were plated as 3 million cells per 10 cm plate the day before transfection, and transfected using 0.5 µg of Flag-ADIPOR1 expression plasmid (Cat # HG14109-NF, Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific).

Techniques: Staining, Western Blot, Derivative Assay, Molecular Weight

Journal: Scientific Reports

Article Title: ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

doi: 10.1038/s41598-018-32579-9

Figure Lengend Snippet: AdipoR1 KO mice and Mfrp rd6 mice have an upregulation of IRBP at 2 weeks of age. ( a ) Western blot of P15 AdipoR1 WT, HET, and KO mouse eyes is shown. α-TUBULIN was used as a loading control. Each lane represents a sample from an individual mouse, n = 7 for WT, n = 8 for HET, n = 8 for KO. ( b ) Western blot of P22 AdipoR1 WT, HET, and KO mouse eyes is shown. α-TUBULIN was used as a loading control. Each lane represents a sample from an individual mouse, n = 3. ( c ) Densitometry quantification of blots from ( a , b ). ( d ) Western blot of P15 Mfrp wt or Mfrp rd6 mouse eyes is shown. α-TUBULIN was used as a loading control. Each lane represents a sample from an individual mouse, n = 7 for Mfrp wt , n = 7 for Mfrp rd6 . ( e ) Western blot of P21 Mfrp wt or Mfrp rd6 mouse eyes is shown. α-TUBULIN was used as a loading control. Each lane represents an eye from a mouse of that genotype and eyes were loaded as pairs from individual animals (5 Mfrp wt and 5 Mfrp rd6 individual animals), n = 10 eyes for Mfrp wt and n = 9 for Mfrp rd6 (one eye was excluded due to low protein levels). ( f ) Densitometry analysis of blots from ( d , e ). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS = not significant. ANOVA (α = 0.05) with Dunnett’s multiple comparisons test (WT set as control) was used in ( c ). Unpaired two tailed t-test was used in ( f ). Error bars represent standard deviation. P values: for part ( c ). IRBP P15 AdipoR1 : WT vs HET - p = 0.9940, WT vs KO - p = 0.0001. IRBP P22 AdipoR1 : WT vs HET - p = 0.0714, WT vs KO - p = 0.2560. For part ( f ). IRBP P15 Mfrp rd6 : p < 0.0001, IRBP P21 Mfrp rd6 p = 0.8777. For all western blots, each membrane was cut and probed separately for the analyzed protein and the loading control.

Article Snippet: To generate exogenous ADIPOR1 expressing cells, HEK293T cells were plated as 3 million cells per 10 cm plate the day before transfection, and transfected using 0.5 µg of Flag-ADIPOR1 expression plasmid (Cat # HG14109-NF, Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific).

Techniques: Western Blot, Two Tailed Test, Standard Deviation

Journal: Scientific Reports

Article Title: ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp rd6 mouse

doi: 10.1038/s41598-018-32579-9

Figure Lengend Snippet: IRBP is mislocalized in the AdipoR1 KO mice. IHC of P15 and P21 mouse eyes of AdipoR1 WT or KO genotypes were stained with the anti-IRBP antibody. IRBP is localized primarily to the OS in WT animals. In AdipoR1 KO retinas IRBP signal appears evenly distributed between the IS and OS. Scale bar = 20 µm. Z-stacks were acquired and the maximum intensity projection is displayed.

Article Snippet: To generate exogenous ADIPOR1 expressing cells, HEK293T cells were plated as 3 million cells per 10 cm plate the day before transfection, and transfected using 0.5 µg of Flag-ADIPOR1 expression plasmid (Cat # HG14109-NF, Sino Biological, Beijing, China) using Lipofectamine 3000 (Thermo Fisher Scientific) and Opti-MEM™ (Thermo Fisher Scientific).

Techniques: Staining