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: For ADIPOR1 protein analysis across different mouse tissues, adult mouse tissue protein lysate from C57BL/6 J mice was used (Cat #: MT-101-C57 skin, MT-102-C57 skeletal muscle, MT-103-C57 adipose tissue, MT-106-C57 eye, MT-313-C57 pancreas, MT-314-C57 liver, MT-201-C57 brain, MT-801-C57 heart, MT-901-C57 kidney - Zyagen, San Diego, California).

Techniques: Expressing, Transfection, CRISPR, Negative Control, Control, Membrane

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: For ADIPOR1 protein analysis across different mouse tissues, adult mouse tissue protein lysate from C57BL/6 J mice was used (Cat #: MT-101-C57 skin, MT-102-C57 skeletal muscle, MT-103-C57 adipose tissue, MT-106-C57 eye, MT-313-C57 pancreas, MT-314-C57 liver, MT-201-C57 brain, MT-801-C57 heart, MT-901-C57 kidney - Zyagen, San Diego, California).

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: For ADIPOR1 protein analysis across different mouse tissues, adult mouse tissue protein lysate from C57BL/6 J mice was used (Cat #: MT-101-C57 skin, MT-102-C57 skeletal muscle, MT-103-C57 adipose tissue, MT-106-C57 eye, MT-313-C57 pancreas, MT-314-C57 liver, MT-201-C57 brain, MT-801-C57 heart, MT-901-C57 kidney - Zyagen, San Diego, California).

Techniques: Expressing, Western Blot, Control, Membrane, 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: For ADIPOR1 protein analysis across different mouse tissues, adult mouse tissue protein lysate from C57BL/6 J mice was used (Cat #: MT-101-C57 skin, MT-102-C57 skeletal muscle, MT-103-C57 adipose tissue, MT-106-C57 eye, MT-313-C57 pancreas, MT-314-C57 liver, MT-201-C57 brain, MT-801-C57 heart, MT-901-C57 kidney - Zyagen, San Diego, California).

Techniques: Western Blot, Control, Membrane, 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: For ADIPOR1 protein analysis across different mouse tissues, adult mouse tissue protein lysate from C57BL/6 J mice was used (Cat #: MT-101-C57 skin, MT-102-C57 skeletal muscle, MT-103-C57 adipose tissue, MT-106-C57 eye, MT-313-C57 pancreas, MT-314-C57 liver, MT-201-C57 brain, MT-801-C57 heart, MT-901-C57 kidney - Zyagen, San Diego, California).

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

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: For ADIPOR1 protein analysis across different mouse tissues, adult mouse tissue protein lysate from C57BL/6 J mice was used (Cat #: MT-101-C57 skin, MT-102-C57 skeletal muscle, MT-103-C57 adipose tissue, MT-106-C57 eye, MT-313-C57 pancreas, MT-314-C57 liver, MT-201-C57 brain, MT-801-C57 heart, MT-901-C57 kidney - Zyagen, San Diego, California).

Techniques: Staining, Western Blot, Control, Membrane, 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: For ADIPOR1 protein analysis across different mouse tissues, adult mouse tissue protein lysate from C57BL/6 J mice was used (Cat #: MT-101-C57 skin, MT-102-C57 skeletal muscle, MT-103-C57 adipose tissue, MT-106-C57 eye, MT-313-C57 pancreas, MT-314-C57 liver, MT-201-C57 brain, MT-801-C57 heart, MT-901-C57 kidney - Zyagen, San Diego, California).

Techniques: Western Blot, Control, Two Tailed Test, Standard Deviation, Membrane

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: For ADIPOR1 protein analysis across different mouse tissues, adult mouse tissue protein lysate from C57BL/6 J mice was used (Cat #: MT-101-C57 skin, MT-102-C57 skeletal muscle, MT-103-C57 adipose tissue, MT-106-C57 eye, MT-313-C57 pancreas, MT-314-C57 liver, MT-201-C57 brain, MT-801-C57 heart, MT-901-C57 kidney - Zyagen, San Diego, California).

Techniques: Staining