- 44-830 - Invitrogen Antibodies SHC1 antibody

Submitted references Input-output behavior of ErbB signaling pathways as revealed by a mass action model trained against dynamic data.

Chen WW, Schoeberl B, Jasper PJ, Niepel M, Nielsen UB, Lauffenburger DA, Sorger PK
Molecular systems biology 2009;5:239

Paxillin-Y118 phosphorylation contributes to the control of Src-induced anchorage-independent growth by FAK and adhesion.

Sachdev S, Bu Y, Gelman IH
BMC cancer 2009 Jan 12;9:12

Insulin enhances growth hormone induction of the MEK/ERK signaling pathway.

Xu J, Keeton AB, Franklin JL, Li X, Venable DY, Frank SJ, Messina JL
The Journal of biological chemistry 2006 Jan 13;281(2):982-92

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Peptide Competition: Extracts prepared from A431 and stimulated with EGF (1-4) or left unstimulated with EGF (5) were resolved by SDS-PAGE on a 10% Tris-glycine gel, and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4C

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (20 µg lysate) of A431 (Lane 1), A431 treated for 10 minutes with 200 ng/mL of EGF (Lane 2), NIH/3T3 (Lane 3) and NIH/3T3 treated for 10 minutes with 200 ng/mL of EGF (lane 4). The blots were probed with Anti-SHC (pY239/pY240) Rabbit Polyclonal Antibody (Product # 44-830, 1:500 dilution) and detected by chemiluminescence Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 51 kDa band corresponding to SHC (pY239/pY240) was observed across the EGF treated cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of A-431 (1), A-431 treated with EGF (200 ng/mL for 10 minutes) (2), A-431 treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) (3), A549 (4), A549 treated with EGF (200 ng/mL for 10 minutes) (5). The blot was probed with Anti-Phospho-SHC (Tyr239, Tyr240) Rabbit Polyclonal Antibody (Product # 44-830, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 51 kDa band corresponding to Phospho-SHC (Tyr239, Tyr240) was detected and observed to increase upon EGF treatment across cell lines tested. Pre-treatment with EGFR-antagonist, Afatinib, resulted in inhibition of Phospho-SHC (Tyr239, Tyr240) in A-431 cell line. Protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane using the wet transfer system. The membrane was probed with the relevant primary and secondary antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed by loading 30 µg of A-431 (Lane 1), A-431 treated with EGF (200 ng/mL for 10 minutes) (Lane 2), A-431 treated with Afatinib followed by EGF (0.5 uM of Afatinib for 6hrs, 200 ng/mL for 10 minutes) (Lane 3), A-431 EGFR KO (Lane 4) A-431 EGFR KO treated with EGF (200 ng/mL for 10 minutes) (Lane 5), A-431 EGFR KO treated with Afatinib followed by EGF (0.5 uM of Afatinib for 6hrs, 200 ng/mL for 10 minutes) (Lane 6) whole cell extracts using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex Protein Standard (Product # LC5800). Proteins were transferred to a nitrocellulose membrane using iBlot® Dry Blotting System (IB21001) and blocked with 5% skim milk for 1 hour at room temperature. The blot was probed with Anti-Phospho-SHC (Tyr239, Tyr240) Rabbit Polyclonal Antibody (Product # 44-830, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 51 kDa band corresponding to Phospho-SHC (Tyr239, Tyr240) was detected and observed to be increased upon EGF treatment in A-431 control cell line (lane1 and 2) and not in EGFR knockout cell line (lane 4 and 5). Pre-treatment with EGFR-antagonist, Afatinib, resulted in inhibition of Phospho-SHC (Tyr239, Tyr240) upon EGF treatment (lane 3 and 6).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of SHC (PYPY239/240) showing staining in the cytoplasm and nucleus of paraffin-embedded human colon carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a SHC (PYPY239/240) Rabbit Polyclonal Antibody (Product # 44-830) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 FAK and adhesion modulate v-Src-induced phosphorylation of various Src substrates . (A) Lysates from adherent cultures of FAK+/+[puro], FAK-/-[puro], FAK+/+[v-Src] and FAK-/-[v-Src] cells were probed either directly by IB for specific phosphorylated form(s) of the Src substrate proteins, total substrate protein levels or GAPDH, or probed for total phosphorylated protein by immunoprecipitating with substrate-specific Ab followed by IB for phosphotyrosine using MAb4G10. (B) Same IB or IP/IB analysis as in panel A using lysates of suspension cultures. Each of these blots is typical of at least duplicate independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Phosphorylation of paxillin poY118 is required for enhanced AIG by FAK-/-[v-Src] cells . ( A ) Left panel- IB analysis of FAK+/+[v-Src] or FAK-/-[v-Src] cell clones (""cl."") stably transfected with empty vector (--) or a GFP-paxillin Y118F -expressing vector, probed with Abs specific for GFP, paxillin poY118 or GAPDH. Aliquots of these cells were analyzed by anchorage-independent growth (top right) or for clonogenic efficiency (bottom right) as described in Materials and Methods. Error bars, S.E. *, P < 0.01. ( B ) A similar analysis as in panel A except on cells stably expressing FLAG-p120 Y228F , with IBs probed for FLAG, p120catenin poY228 or GAPDH. Note that there is no statistical difference in the p120catenin Y228F -mediated decrease in AIG between the FAK+/+[v-Src] and FAK-/-[v-Src] cells. ( C ) A similar analysis as in panel A except on cells stably expressing GST-Shc Y239/240F , with IBs probed for GST-tag, Shc poY239/240 , or GAPDH.

44-830

Antibody data