Anti-Human ErbB2 Recombinant Antibody (Trastuzumab) (CAT#: TAB-005)

Figure 1 t-DARPP and ERBB2 are significantly overexpressed in adenocarcinomas.

left, cell viability of OE19 and OE33 cells in response to trastuzumab treatment was evaluated by Trypan blue staining. OE19 cells were 2-fold more sensitive to trastuzumab than OE33 cells (P < 0.001). D, right, Western blot analysis shows higher protein expression of ERBB2 in OE19 cells than OE33 cells. In contrast, t-DARPP expression was undetectable in OE19 cells but highly expressed in OE33 cells.

Hong, J., Katsha, A., Lu, P., Shyr, Y., Belkhiri, A., & El-Rifai, W. (2012). Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma. Cancer research.

Figure 2 t-DARPP expression blocks trastuzumab-induced apoptosis.

A, apoptosis in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) recombinant adenoviruses after treatment with vehicle or trastuzumab (20 μg/mL) for 48 hours was determined by Annexin-V/PI staining and FACS analysis. B, Western blot analysis of caspase-3, cleaved caspase-3, and t-DARPP proteins in OE19 cells infected with control or t-DARPP adenoviruses following treatments as described in A. C, apoptosis in parental and trastuzumab-resistant OE19 cells after treatment with vehicle or trastuzumab (20 μg/mL) for 48 hours was evaluated by Annexin-V/PI staining and FACS analysis. D, Western blot analysis of caspase-3, cleaved caspase-3, and t-DARPP proteins in parental and trastuzumab-resistant OE19 cells after treatments as described in C. These data indicate that endogenous and exogenous t-DARPP expression counteracted trastuzumab-induced apoptosis in OE19 cells.

Hong, J., Katsha, A., Lu, P., Shyr, Y., Belkhiri, A., & El-Rifai, W. (2012). Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma. Cancer research.

Figure 3 t-DARPP associates with ERBB2 and interferes with trastuzumab/ERBB2 protein interaction.

A, Western blot analysis of coimmunoprecipitated exogenous t-DARPP and endogenous ERBB2 proteins with M2-flag or trastuzumab antibodies in OE19 cells infected with t-DARPP-flag adenovirus (10 MOI). These data show protein association of ERBB2 with t-DARPP. B, Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in OE19 cells infected with control (10 MOI) or t-DARPP (10 MOI) adenoviruses. Pulled-down ERBB2 band intensity was depicted as a ratio relative to input ERBB2 protein. The results show that exogenous t-DARPP expression blocked binding of trastuzumab to ERBB2 receptor relative to control. C, Western blot analysis of immunoprecipitated endogenous ERBB2 protein with trastuzumab antibody in parental or trastuzumab-resistant OE19 cells. The band intensity of immunoprecipitated ERBB2 protein was shown as a ratio relative to input ERBB2. These data indicate that endogenous t-DARPP expression in trastuzumab-resistant cells was associated with a significant decrease in trastuzumab/ERBB2 protein interaction relative to control.

Hong, J., Katsha, A., Lu, P., Shyr, Y., Belkhiri, A., & El-Rifai, W. (2012). Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma. Cancer research.

Figure 4 t-DARPP overexpression promotes tumor growth and blocks response to trastuzumab treatment in vivo.

The H&E staining at the end of trastuzumab treatment shows effectively diminished control tumors leaving necrotic and fibrotic lesions (left), whereas t-DARPP tumors were unaffected (right).

Hong, J., Katsha, A., Lu, P., Shyr, Y., Belkhiri, A., & El-Rifai, W. (2012). Regulation of ERBB2 receptor by t-DARPP mediates trastuzumab resistance in human esophageal adenocarcinoma. Cancer research.

Figure 5 Enhanced internalization of ErbB2 in caveolin-1 expressing SKBR-3 cells.

(A) SKBR-3/Cav-1 cells were incubated with 1 μM of EC-eGFP at 37°C for 5 min and internalization was assessed using anti-ErbB2 monoclonal antibody. Cell surface binding of EC-eGFP (green) with ErbB2 (red) was shown in the merged picture (yellow). (B) enhanced endocytosis of Ec-eGFP (green) and Intracellular co-localization with ErbB2 (red) was observed after 15 min of incubation at 37°C. (C) Parental SKBR-3 cells incubated with EC-eGFP for 15 min at 37°C were processed for endocytosis analysis as above using anti ErbB2 antibody (red). (D) SKBR-3/Cav-1 cells incubated with Trastuzumab (green) for 5 min at 37°C were processed and stained with anti-caveolin-1 antibody (blue); cell surface binding of Trastuzumab with caveolin-1 was shown (opaque) in the merged image. (E) Internalization and localization of Trastuzumab (green) with caveolin-1 (blue) in SKBR-3/Cav-1 cells after 15 min of stimulation at 37°C. (F) Parental SKBR-3 cells incubated similarly with Trastuzumab for 15 min were processed for endocytosis analysis as detailed in the material and methods section. Scale bar: 20 μm.

Sekhar, S. C., Kasai, T., Satoh, A., Shigehiro, T., Mizutani, A., Murakami, H., ... & Seno, M. (2013). Identification of caveolin-1 as a potential causative factor in the generation of trastuzumab resistance in breast cancer cells. Journal of Cancer, 4(5), 391.

Figure 6 Ligand induced ErbB2 internalization after EC-eGFP and Trastuzumab treatment.

SKBR-3 cells were surface biotinylated to monitor internalization. (A) Biotinylated caveolin-1 expressing SKBR-3 cells were stimulated with 1 μM EC-eGFP and Trastuzumab for 15, 30 and 60 min at 37°C, 4°C or left untreated for 1 h at 37°C and 4°C. Densitometry was performed using Image J software. (B) Wild type SKBR-3 cells were incubated with Ec-eGFP and Trastuzumab as described above for 15-60 min. Cell lysates prepared from treated samples were then pull-downed with avidin agarose and subjected to western blotting using an anti-ErbB2 antibody. Endocytosed transferrin receptor was also monitored simultaneously as internal control. The results are expressed as the mean SD of three individual experiments. Since there was no significant internalization observed in parental SKBR-3 cells, densitometry was performed only for caveolin-1 expressing SKBR-3 cells.

Sekhar, S. C., Kasai, T., Satoh, A., Shigehiro, T., Mizutani, A., Murakami, H., ... & Seno, M. (2013). Identification of caveolin-1 as a potential causative factor in the generation of trastuzumab resistance in breast cancer cells. Journal of Cancer, 4(5), 391.

Figure 7 Fc receptor mediated ADCC in SKBR-3 cells.

ADCC activity mediated by Trastuzumab and EC-Fc against cell surface ErbB2 in SKBR-3, SKBR-3/Cav-1 and SKOV-3 cells was measured using human PBMC's as effector cells at an effector: target cell ratio of (A) 25:1 (B) 50:1 and (C) 100:1, with standard LDH assay as described in materials and methods. Data are expressed as the mean of (±) SD (n=3). Student's t- test (two tailed) was used to compare the ADCC response in SKBR-3/Cav-1 and SKOV-3 with parental SKBR-3. Differences were statistically significant at P < 0.05.

Sekhar, S. C., Kasai, T., Satoh, A., Shigehiro, T., Mizutani, A., Murakami, H., ... & Seno, M. (2013). Identification of caveolin-1 as a potential causative factor in the generation of trastuzumab resistance in breast cancer cells. Journal of Cancer, 4(5), 391.

Figure 1 Anti-ErbB2 Antibody (TAB-005) in WB

Western blot analysis of TAB-005 was performed with human ErbB2 protein.

Figure 2 Anti-ErbB2 Antibody (TAB-005) in ELISA

ELISA analysis of TAB-005 was performed by coating with human ErbB2 protein.

Figure 3 Anti-ErbB2 Antibody (TAB-005) in Dot Blot

Dot Blot analysis of TAB-005 was performed by coating with human ErbB2 protein.

Figure 4 Anti-Human ErbB2 Recombinant Antibody (Trastuzumab) (TAB-005) in HPLC

The purity of TAB-005 was greater than 95% as determined by SEC-HPLC.

Figure 5 Anti-Human ErbB2 Recombinant Antibody (Trastuzumab) (TAB-005) in SDS-PAGE

SDS-PAGE analysis of TAB-005 in reduced conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.