- PA1-41220 - Invitrogen Antibodies RTN4 antibody

Submitted references REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects.

Lee SH, Hadipour-Lakmehsari S, Murthy HR, Gibb N, Miyake T, Teng ACT, Cosme J, Yu JC, Moon M, Lim S, Wong V, Liu P, Billia F, Fernandez-Gonzalez R, Stagljar I, Sharma P, Kislinger T, Scott IC, Gramolini AO
Nature communications 2020 Feb 19;11(1):965

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Nogo-A/B in A) human, B) mouse, and C) rat brain tissue lysate. Samples were incubated in Nogo-A/B polyclonal antibody (Product # PA1-41220) using a dilution of 1:2000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Nogo-A/B in 0.5 mg/mL Human Brain lysate. Samples were incubated in Nogo-A/B polyclonal (Product # PA1-41220). This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot was performed using Anti-Nogo-A/B Polyclonal Antibody (Product # PA1-41220) and a 200 kDa band corresponding to Reticulon-4 was observed across all the cell lines and tissue lysates tested except for MCF7 as reported across protein expression databases. Membrane enriched extracts (30 µg lysate) of U-87 MG (Lane 1), Daudi (Lane 2), MCF7 (Lane 3), Mouse Brain (Lane 4), Rat Brain (Lane 5), Mouse Cerebellum (Lane 6), Rat Cerebellum (Lane 7), Mouse Heart (Lane 8), Rat Heart (Lane 9) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:500 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Reticulon-4 was performed using 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Nogo-A/B Polyclonal Antibody (Product # PA1-41220) at (1:100 dilution) in 0.1% BSA, incubated at 4-degree Celsius overnight, and labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical analysis of Nogo-A/B in human testis. Samples were incubated in Nogo-A/B polyclonal antibody (Product # PA1-41220) using a dilution of 1:100.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 6 Mass spectrometry analysis identifies REEP5 interactions with known cardiac SR shaping proteins. a Immunoblot analysis of nickel-His purification of CnVA-REEP5 from transfected HEK293 cells shows stable expression of CnVA-REEP5 and successful immunoprecipitation of CnVA-REEP5 monomer (~34 kDa) and dimer (~68 kDa). b Identification of known SR/ER-shaping proteins as REEP5 interacting proteins by mass spectrometry analysis. Average precursor MS1 peak areas (peptide m/z signal) as defined by iBAQ (intensity based absolute quantification) are shown, n = 3 independent mass spectrometry runs. To calculate fold change, average values (n.d.--not detected) were inputted with a value of 10. RTN reticulon, ATL atlastin, CKAP4 cytoskeleton-associated protein 4, ACTC1 alpha cardiac muscle actin 1, MYL6 myosin light chain 6, GAPDH glyceraldehyde-3-phosphate dehydrogenase. Asterisks indicate a statistically significant p value in a Tukey's multiple comparison analysis where * p < 0.05. c RNASeq analysis of the RTN, ATL families of proteins, and CKAP4 in human fetal heart and adult heart tissue data using data from Human Protein Atlas. d Immunoblot analysis of nickel-His REEP5 immunoprecipitation lysates for RTN4/Nogo-A/B, ATL3, and CKAP4, n = 3. e HEK293 c e lls were transfected with myc-tagged Nogo-A and Nogo-B plasmids and detected with myc and alpha-tubulin antibodies. f Co-immunoprecipitation assay of cotransfected HEK293 cells with anti-REEP5 antibody (left panel) and anti-

PA1-41220

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