- PA5-28622 - Invitrogen Antibodies CRKL antibody

Submitted references The Effector TepP Mediates Recruitment and Activation of Phosphoinositide 3-Kinase on Early Chlamydia trachomatis Vacuoles.

Carpenter V, Chen YS, Dolat L, Valdivia RH
mSphere 2017 Jul-Aug;2(4)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of CrkL using 50 µg of mouse brain lysate. Samples were loaded onto a 12% SDS-PAGE gel and probed with a CrkL polyclonal antibody (Product # PA5-28622) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of CrkL using 30µg of A) 293T (B) A431 (C) H1299 (D) HeLa S3 (E) HepG2 (F) MOLT4 and G) Raji lysate. Samples were loaded onto a 12% SDS-PAGE gel and probed with a CrkL polyclonal antibody (Product # PA5-28622) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of CrkL in Mouse tissue extract (50 µg). Samples was separated by 12% SDS-PAGE and the membrane was probed with CrkL Polyclonal antibody (Product # PA5-28622) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of CrkL was performed by separating 30 µg of various whole cell extracts by 12% SDS-PAGE. Proteins were transferred to a membrane and probed with a CrkL Polyclonal Antibody (Product # PA5-28622) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using CrkL Polyclonal Antibody (Product # PA5-28622). Mouse tissue extract (50 µg) was separated by 12% SDS-PAGE, and the membrane was blotted with CrkL Polyclonal Antibody (Product # PA5-28622) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of CRKL was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR903833_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of CRKL was performed by loading 50 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) andHeLa CRKL KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-CrkL Polyclonal Antibody (Product # PA5-28622, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to CRKL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Crk-like protein was achieved by transfecting SH-SY5Y with Crk-like protein specific siRNAs (Silencer® select Product # S3523, S3524). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Crk-like protein untransfected cells (lane 1), non-targeting scrambled siRNA transfected cells (lane 2) and knockdown cells (lane 3). The blot was probed with CrkL Polyclonal Antibody (Product # PA5-28622, 1:1000 dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 Crk-like protein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-CrkL Polyclonal Antibody (Product # PA5-28622) and a 39kDa band corresponding to Crk-like protein was observed across cell lines tested. Whole cell extracts (30 µg lysate) of SH-SY5Y (Lane 1), Hep G2 (Lane 2), HeLa (Lane 3), HEK-293 (Lane 4), PC-12 (Lane 5), Mouse Brain (Lane 6) 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 (1:1000 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 Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of CrkL was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: CrkL Polyclonal Antibody (Product # PA5-28622) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Crk-like protein was performed using 70% confluent log phase SH-SY5Y 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 CrkL Polyclonal Antibody (Product # PA5-28622) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (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 Cytoplasm 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: Immunohistochemistry (Paraffin) analysis of CrkL was performed in paraffin-embedded human breast carcinoma tissue using CrkL Polyclonal Antibody (Product # PA5-28622) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CrkL Polyclonal Antibody detects CrkL protein at cytosol and nucleus on human ovarian carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human ovarian carcinoma. CrkL Polyclonal Antibody (Product # PA5-28622) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CrkL Polyclonal Antibody detects CrkL protein at cytosol on human colon carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human colon carcinoma. CrkL Polyclonal Antibody (Product # PA5-28622) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of CrkL was performed in 293T whole cell extracts using 5 µg of CrkL Polyclonal Antibody (Product # PA5-28622). Samples were transferred to a membrane and probed with CrkL Polyclonal Antibody as a primary antibody and an HRP-conjugated anti-Rabbit IgG was used as a secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 2 TepP is required for C. trachomatis replication in A2EN cells and to recruit CrkL and PI3K to nascent inclusions. (A to D) Phenotypic characterization of a C. trachomatis Delta tepP :: bla insertional mutant. (A) Immunoblot analysis of TepP expression in HeLa cells infected for 24 h with the indicated strains. Slc1, loading control. (B) A2EN cells were infected with CTL2 or Delta tepP :: bla bacteria, and the levels of IFIT1 (black) and IFIT2 (gray) transcripts from the strains described in the panel A legend were assessed by quantitative RT-PCR. DNA transfection was used as a positive control for the induction of IFIT genes. (C) Time course of appearance of major tyrosine-phosphorylated (p-Tyr) proteins in A2EN cell infected with CTL2 or Delta tepP :: bla insertional mutants or left uninfected (Uninf.). MOMP, bacterial major outer membrane protein (arrowhead); Tub, tubulin. (D) The replication potential of Delta tepP :: bla mutants was assessed in HeLa and A2EN cells by the generation of inclusion-forming units (IFU). (E and F) Subcellular localization of CrkL, PI3K (p110alpha), and GSK-3beta in Chlamydia -infected cells. A2EN cells were infected with CTL2 or Delta tepP :: bla bacteria for 4 h and immunostained by anti-MOMP (green), anti-CrkL, anti-p110alpha, or anti-GSK-3beta (red). Hoechst (blue) was used to detect DNA. Quantification of Chlamydia (MOMP) and p110alpha, CrkL, and GSK-3beta colocalization was performed on a single-cell basis ( n = 20 and 30 cells/repli

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 3 TepP is phosphorylated by Src kinases. (A) In vitro phosphorylation reactions were performed by incubating recombinant 6xHis-TepP with ATP and cell lysates derived from the indicated cell lines. The degree of TepP phosphorylation was assessed by immunoblotting with anti-pY antibodies after reisolation of TepP on nickel beads. TepP was phosphorylated under all conditions except after incubation with SYF cell lysates and the buffer-only control. (B) TepP-FLAG immunoprecipitations from cell lines infected with CTLM062G1 expressing TepP-FLAG. TepP phosphorylation was assessed after IP with anti-FLAG antibodies and immunoblotting with anti-pY antibodies. Note the decreased levels of phosphorylation in Src, Yes, and Fyn-deficient cells. (C) Mouse embryo fibroblast (MEF), SYF, and SYF-plus-c-Src cell lines were infected for 4 h with C. trachomatis at an MOI of 20, fixed, and stained. Infected cells were immunostained by anti-MOMP (green), anti-CrkL, or anti-p110alpha (red) antibodies.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 4 PI3K and CrkL independently associate with early inclusions. (A) HeLa cells stably expressing Cas9 (HeLa-Cas) were transduced with sgRNAs specific to PIK3KCA , CRKL , and CRKI-II to generate gene-edited cell lines lacking expression of the respective target proteins. Loss of protein expression for each cell line was verified by immunoblot analysis using anti-p110alpha, anti-CrkL, and anti-Crk I/II antibodies. Tubulin levels were used as positive controls. There was a low level of cross-reactivity between CrkL and CrkII antibodies. (B) CrkL and p110alpha are recruited to early inclusions formed in PI3KCA and CRKL gene-edited knockout cells (KO), respectively. HeLa-Cas cells and their edited derivatives were infected with CTL2 for 4 h, fixed, and immunostained with anti-MOMP and anti-p110alpha or anti-CrkL (red) antibodies. Host and bacterial DNA were detected with Hoechst (blue). (C) Replication of WT and TepP-deficient C. trachomatis in PI3KCA , CRKL , and CRK gene-edited knockout cells (KO).

PA5-28622

Antibody data