MA1-813

antibody from Invitrogen Antibodies

Targeting: RLBP1 CRALBP

Provider product page for MA1-813

Western blot

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [31]
Comments [0]
Validations
Immunohistochemistry [1]
Other assay [13]

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Product number: MA1-813 - Provider product page
Provider: Invitrogen Antibodies
Product name: CRALBP Monoclonal Antibody (B2)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA1-813 detects CRALBP protein in bovine, human, mouse, non-human primate and rat samples. MA1-813 has successfully been used in Western blot, immunohistochemistry, immunoprecipitation, and immunofluorescence procedures. By Western blot, this antibody detects a 37 kDa protein representing human recombinant CRALBP. The MA1-813 immunogen is purified human recombinant CRALBP.
Reactivity: Human, Mouse, Rat, Bovine
Host: Mouse
Isotype: IgG
Antibody clone number: B2
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

RLBP1 protein structure - MA1-813 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 1 A Mesenchymal-Epithelial Transition with temporal FOXM1 expression during RPE culture. A. Immunocytochemistry was performed for CRALBP, ZO1, alphaSMA and Ki67 at Day 0 (D0), Day 3 (D3) and Day 35 (D35). Arrowheads point towards Ki67 positive nuclei. B. Microarray heatmap of the expression profiles of the top 250 genes, ranked by the significance of their expression changes, over time in culture. Raw expression data are mean centred and scaled to unit variance prior to clustering. A schematic of the scaled expression is shown on the right where individual gene profiles are in light grey and the mean expression profile is shown in black. C. Microarray heatmap showing transcript expression for a panel of representative markers over a timecourse of RPE culture. D. Immunocytochemistry for FOXM1 at Day 2 and Day 14 of RPE culture. Arrowheads point towards FOXM1 positive nuclei. E. Quantification of immunocytochemistry showing percentage of nuclei staining positive for FOXM1 over time. Bars represent Mean +- SD (n = 3). F. Expression of FOXM1 transcript measured using qPCR (relative to housekeeping genes ACTB and GAPDH ) in iPSC derived RPE, human foetal RPE and ARPE19 cells over time. Bars represent Mean +- SD (n = 3)

Supportive validation

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Experimental details: Figure 1 Patterns of eGFP expression mediated by reporter vectors in mouse and monkey retinas. ( a ) Following dissection of the neural retina from a mouse eye injected with scAAV8p RLBP1 (short)-eGFP, the remaining eye cup was fixed, flat-mounted and imaged. The photograph shows the large geographic regions of RPE that express eGFP. ( b-p ) Neural retina was fixed, sectioned and costained with cell-specific antibody markers. ( b-d ) Images of a retina transduced by the scAAV8-p RLBP1 (short)-eGFP vector showing eGFP, CRALBP, and the merger of eGFP and CRALBP signals demonstrate eGFP expression in CRALBP-expressing Muller cells spanning the retina from the external limiting membrane to the end feet in the inner limiting membrane; ( e-h ) eGFP, vimentin, eGFP+vimentin merged, and eGFP+GFAP merged indicate that eGFP is coexpressed with vimentin and GFAP in Muller cell processes of the inner nuclear layer and the inner limiting membrane where vimentin and GFAP are in intermediate filaments of Muller cells. ( i-k ) Images of eGFP, blue opsin, and eGFP+blue opsin merged reveal that eGFP does not colocalize with cone photoreceptors that express blue opsin, and ( m-o ) images of eGFP, green opsin, and eGFP+green opsin merged show that eGFP does not colocalize with cone photoreceptors that express green opsin. As examples of off-target photoreceptor expression, images from a ( l ) merger of images of eGFP+blue opsin expression from a retina injected with ssAAV8-p BEST -eGFP injected

Supportive validation

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Experimental details: Fig 6 BMP/Wnt signalling is required for MET. A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10muM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett's multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10muM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett's multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P

Supportive validation

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Experimental details: Figure 2 MITF is required and sufficient to induce RLBP1 and RDH5 expression in ARPE-19 cells. ( A ) The expression of RLBP1 and RDH5 in ARPE-19 cells transfected with scramble siRNA (si-C) or various concentrations of MITF siRNA (si-MITF). Increased si-MITF concentrations resulted in enhanced MITF knockdown. The expression of the MITF target gene TYR was reduced concomitantly with the reduction of MITF . With the reduction in MITF expression, the expression of RLBP1 and RDH5 was also decreased. ( B ) Protein expression of RLBP1 and RDH5 was decreased when MITF was knocked-down in ARPE-19 cells. TUBULIN was used as an internal protein loading control. ( C ) The RNA and protein levels of RLBP1 and RDH5 in EGFP- and MITF- overexpressing ARPE-19. Both RNA and protein expression of RLBP1 and RDH5 were increased when MITF was overexpressed in ARPE19 cells compared to overexpression of EGFP . The experiments were performed in triplicate and the error bars represent the S.E. *indicates P < 0.05.

Supportive validation

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Experimental details: Figure 4 The differentiation of 3D-RPE cells ( A-C ) RT-qPCR analysis of expression of CRALBP, BEST1 and RPE65 in 3D-RPE cells versus time-matched SD-RPE cells at day 40, day 60 and day 100 after differentiation respectively (bars: mean + s.d.; n = 3). All samples were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Single asterisk (*) indicated p value < 0.05 and double asterisk (**) indicated p value < 0.01. ( D-F ) Immunostaining of CRALBP and BEST1 of 3D-RPE and SD-RPE cells at day 40, day 60 and day 100 after differentiation respectively. Z-stack fluorescent images with cross-section side views were displayed to show BEST1 location.

Supportive validation

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Experimental details: Figure 3 Characterization of the Rlbp1 -/- mouse. ( a ) The horizontal bar is a schematic representation of the Rlbp 1 targeted deletion in the Lexicon TF0133 mouse line. Primers used for genotyping are depicted as arrows (P-9, P-3, and P-14). ( b ) PCR analysis of DNA from Rlbp1 -/- , Rlbp1 +/- , and Rlbp1 +/+ mice using primers P-9, P-3, and P-14 show the expected 854 bp amplicon of the WT allele and the 358 bp amplicon of the allele carrying the deletion. ( c,d ) Paraffin sections of Rlbp1 +/+ ( c ) and Rlbp1 -/- ( d ) mouse eyes stained with an antibody against CRALBP. ( e,f ) Hematoxylin and eosin stained paraffin sections of 16-month-old Rlbp1 +/+ ( e ) and Rlbp1 -/- ( f ) mouse eyes. ( g ) Lysates of neural retina from uninjected Rlbp1 -/- (KO uninj.), Rlbp1 -/- injected with 1 x 10 9 vg/eye of scAAV8-p RLBP1 (short)-h RLBP1 (KO, vector inj.), and uninjected Rlbp1 +/+ (WT uninj.) mice were analyzed by western blot using a primary antibody against CRALBP. Arrow, end feet of Muller cells; arrow head, Muller cell processes; diamond, cell bodies of inner nuclear layer; asterisk, RPE; scale bars, 50 um in c+d and 100 um in e+f .

Supportive validation

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Experimental details: Fig. 6 Development of NR2E1 MiniPromoters for Muller glia cells. A Bioinformatic design of first-generation Ple264, cutdown to produce second-generation Ple315, and redesign to produce second-generation Ple316. ENCODE segments that potentially regulate the expression of the NR2E1 gene are highlighted horizontally. Vertically highlighted genomic regions correspond to their color-matched segments included in the MiniPromoter designs and are numbered as promoter(s) (P) and regulatory region(s) (RR). B Postnatal day 4 intravenous injection of Ple316-EmGFP, harvested 4 weeks later, led to robust expression with cell bodies in the inner nuclear layer (INL) and processes that span the retina. Scale bar, 100 um. (For ubiquitous smCBA promoter see Fig. S1A .) C Adult intravitreal injection of Ple316-EmGFP, harvested 4 weeks later, was quantified for epifluorescence intensity in CRALBP-positive cells, and showed no significant difference when compared to smCBA. (For smCBA see Fig. S1D ). D Adult intravitreal injection of Ple316-EmGFP, harvested 4 weeks later, led to robust expression in the Muller glia cells, as indicated by co-staining with Muller glia cell marker CRALBP. Scale bar, 20 um. (For smCBA see Fig. S1D .) EmGFP emerald green fluorescent protein, GCL ganglion cell layer, IPL inner plexiform layer, N number of cells counted, ONL outer nuclear layer, OPL outer plexiform layer, TF transcription factor, TSS transcription start site. Green, anti-GFP; blue, Hoechst; red, anti-CRAL

Supportive validation

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Experimental details: Figure 4 Coimmunostaining of P3 foveal glial cells with the Muller cell marker CRALBP, GFAP, and vimentin. Coimmunostaining of foveal cryosections from P3 with cellular retinaldehyde-binding protein (CRALBP), which is a specific marker of Muller cells in humans, glial fibrillar acidic protein (GFAP), and vimentin (VIM), which stains all glial origin cells, shows that the GFAP-positive cells at the roof of the foveal pit ( A , B , C , D , and inset) are stained with vimentin ( G , H , and inset) but do not express CRALBP ( E , F , and inset). I : Costaining with GFAP and vimentin shows that the GFAP-positive cells at the roof of the pit (arrow) are faintly stained with vimentin and are distinct from the vimentin-positive Muller glial cell extensions (red arrow). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; HL, Henle's fiber layer; OLM, outer limiting membrane.

Supportive validation

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Experimental details: Figure 3 Visual cycle proteins were abundantly expressed in the RPE flatmounts during the first 3 days in culture, but gradually decreased over time. RPE flatmounts obtained from 2- and 9-month-old wild-type C57BL/6J mice were cultured in complete medium for up to 7 days. RPE flatmounts treated with H 2 O 2 were cultured for 16 h. ( A ) Protein lysates were prepared from the RPE flatmounts and subjected to western blotting analysis to detect the levels of visual cycle proteins RPE65 and CRALBP. Statistical analysis was performed using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 3. ( B ) RPE65 and CRALBP protein levels were also measured by immunofluorescence. Data from both the western blotting and immunofluorescence showed the gradual reduction in the two proteins in RPE flatmounts over time. Scale bar = 20 mum.

Supportive validation

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Experimental details: Figure 1. Immunophenotypic characterization of mouse MG cell line RG17. (A) Phase contrast images and immunofluorescence staining of primary MG cells and passage 20 RG17 cells. RG17 demonstrate typical MG morphology in culture and express many well-characterized MG markers. Scale bar, 50 um and applies to all immunofluorescence images. (B) Expression of characteristic MG genes in primary MG and RG17 cells (passages 20-25). The geometric means and geometric standard deviations ( N = 3 independent cultures) are graphed. An unpaired t -test was applied with a P value of

Supportive validation

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Experimental details: Figure 2. Immunophenotypic characterization of X laevis MG cell line XG69. (A) Phase contrast images and immunofluorescence staining of primary MG and XG69 cell line at passage 20. (B) Expression of MG genes in primary MG and XG69 cells (passages 20-25). The geometric means and geometric standard deviations ( N = 3 independent cultures) are graphed. An unpaired t -test was applied with a P value of