- PA5-46931 - Invitrogen Antibodies CLU antibody

Submitted references Isolation, culture, and cryosectioning of primary porcine retinal pigment epithelium on transwell cell culture inserts.

Hood EMS, Curcio CA, Lipinski D
STAR protocols 2022 Dec 16;3(4):101758

Sterol O-acyltransferase 2 chaperoned by apolipoprotein J facilitates hepatic lipid accumulation following viral and nutrient stresses.

Sun HY, Chen TY, Tan YC, Wang CH, Young KC
Communications biology 2021 May 12;4(1):564

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Apolipoprotein J was achieved by transfecting HeLa cells with Apolipoprotein J specific siRNAs (Silencer® select Product # s3156, s3158). Western blot analysis (Fig. a) was performed using whole cell extracts from the HeLa knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Apolipoprotein J Goat Polyclonal Antibody (Product # PA5-46931, 0.1 µg/mL) and Rabbit anti-Goat IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Apolipoprotein J.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Apolipoprotein J Polyclonal Antibody (Product # PA5-46931) and 38-75 kDa bands corresponding to Apolipoprotein J was observed across cell lines tested except Daudi, K-562 and 3T3-L1 fibroblasts. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), Daudi (Lane 2), K-562 (Lane 3), 3T3-L1 (Lane 4) and 3T3-L1 differentiated to Adipocytes (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 (0.1 µg/mL) and detected by chemiluminescence with Rabbit anti-Goat IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 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: Western blot was performed using Anti-Apolipoprotein J Polyclonal Antibody (Product # PA5-46931) and 32-75 kDa bands corresponding to Apolipoprotein J was observed across tissues tested. Whole cell extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2), Mouse Liver (Lane 3) and Mouse Brown Fat (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 (0.1 µg/mL) and detected by chemiluminescence with Rabbit anti-Goat IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 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 Apolipoprotein J was performed using HeLa 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 1 hour at room temperature. The cells were labeled with Apolipoprotein J Goat Polyclonal Antibody (Product # PA5-46931) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Rabbit anti-Goat IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27012) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green) in HeLa cells. 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). Panel d represents the merged image of HeLa cells showing cytoplasmic and nuclear 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)
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Experimental details: Fig. 1 ApoJ participated in HCV-induced lipid accumulation. a Representative images of LDs in Huh7.5 cells with mock or HCV infection at day 6 post-infection. Scale bar, 5 mum. b The diameters of LDs ( n > 2000) were quantified with ImageJ software. The data were collected from three independent experiments ( n >= 8). Quantification of the intracellular contents of TC ( c ), CE/FC ratio ( d ), and TG ( e ) with acute or chronic HCV infections ( n >= 6). The hepatic TC level ( f ) and CE/FC ratio ( g ) in wild type ( n = 5) and HCV coreTg mice ( n = 7). h Protein levels of ApoJ and HCV-NS3 in Huh7.5 cells with acute or chronic HCV infections. The right panel: ApoJ intensities ( n = 3); psApoJ, ApoJ precursor. i Protein levels of ApoJ and HCV core in control and ApoJ-silenced Huh7.5 cells inoculated with HCV (MOI = 0.01) at day 6 post-infection. j TC, CE/FC ratio and TG contents and with HCV infection in the control and ApoJ-silenced Huh7.5 cells at day 6 post-infection. All results are presented as the mean +- SEM, and asterisks indicate statistical significance ( p < 0.05). k Images of LDs visualized by CholEsteryl BODIPY(tm) 542/563 C 11 tracer with MOI = 0.01 or 0.5 at post-infection day 3. Scale bar, 50 mum. Uncropped blots of h and thereafter are displayed in Supplementary Figs. 15 - 30 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 FFA-induced hepatic lipid accumulation depended on ApoJ. The cellular contents of TC and TG were quantified in Huh7 cells treated with PA ( a ) or OA ( b ) for 24 h ( n >= 5), CE/FC ratio of PA and OA treated cells ( c ) ( n >= 6), and ApoJ expression in Huh7 cells treated with PA ( d ) or OA ( e ). The lower panel: ApoJ intensities ( n = 3); psApoJ, ApoJ precursor. f Representative images of LDs stained with BODIPY493/503, and g quantification of lipid contents in Huh7.5 cells bearing Luc or ApoJ shRNA treated with PA or OA for 24 h. Scale bar, 100 mum in f . h Quantification of lipid contents in the cells transfected with ApoJ expressing plasmid or control vector. All results are normalized to the respective untreated control and presented as the mean +- SEM ( n >= 5), and asterisks indicate statistical significance ( p < 0.05). Representative IFA images of SOAT2 (red) and Golgi (TGN38, green) ( i ); SOAT2 and ApoJ (green) ( j ) in PA- or OA-treated cells were analysed by Coloc2 plugin and FVW31S software as in Fig. 2d . Scale bar, 5 mum. Asterisks indicate statistical significance ( p < 0.05). k The PA- (200 muM) or OA- (800 muM) treated Huh7 cells were fed with NBD-Chol, and the SOAT activity was evaluated and expressed as the mean +- SEM ( n = 6). l The protein level of SOAT2 in Huh7 cells treated with PA or OA for 24 h. The lower panel: SOAT2 intensities ( n >= 2). m Huh7 cells were treated with 800 muM OA for 24 h. The cell lysate was immunoprecipitated with ant

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5 ApoJ co-localized with SOAT2 by recognizing the IDR region. The IDR regions of SOAT1 (UniProt ID: P35610, a ) and SOAT2 (UniProt ID: O75908, b ) were predicted by D 2 P 2 ( http://d2p2.pro/search ) and IUPred2A ( https://iupred2a.elte.hu/ ) algorithms, respectively. Huh7 cells were transfected with SOAT1-DsRed ( c , the left panels), IDR-truncated SOAT1-DsRed (DeltaSOAT1-DsRed, c , the right panels); SOAT2-DsRed ( d , the left panels), and IDR-truncated SOAT2-DsRed (DeltaSOAT2-DsRed, d the right panels), followed by administration of PA (100 muM) or OA (400 muM) for 24 h, and visualized with confocal microscopy. The fluorescence intensity was analysed by FVW31S software. The Mander''s coefficient was analysed by ImageJ with the Coloc2 plugin from ImageJ/Fiji shown above. Scale bar, 5 mum. Huh7 cells were transfected with plasmids encoding SOAT2-DsRed ( e ) or DeltaSOAT2-DsRed ( f ). The cell lysates were immunoprecipitated with antibody against ApoJ and examined by western blot analysis recognizing DsRed and apoJ.

PA5-46931

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