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. 2017 Jul 28;8(40):68493–68516. doi: 10.18632/oncotarget.19663

Figure 6. Immunoblot studies on SIL1 mutant lymphoblastoid cells and tissue.

Figure 6

(A) BiP-immunoprecipitation in control and MSS-patient derived lymphoblastoid cells and subsequent analyses of amont of BiP-bound GRP170 reveals a higher amount of BiP-associated GRP170 in the patient cell pool compared to the controls. (B) Immunoblot-based verification of protoemic findings for paradigmatic proteins (SIL1, GRP94, RAB11-FIP1, PHGDH, ACTA, CALM, α-Syn & SELH) utilizing an idependent batch of control and MSS-patient derived LCs. Investigation confirmed a decrease for SIL1, GRP94, PHGDH, ACTA, & CALM as well as an increase for RAB11-FIP1,α-Syn & SELH. GAPDH has been used to demonstrate equal protein loading. (C) Investigation of ER-stress responsiveness of the above mentioned paradigmatic proteins confirmed the known up-regulation of SIL1 and GRP94 upon presence of thapsigargin-induced ER-stress and moreover revealed a similar effect for RAB11-FIP11 (but not further elevated in patient cells upon thapsigargin-treatment), PHGDH and SELH. Increase of α-Syn upon thapsigargin-treatment is most likely in agreement with the build-up of protein aggregates. GAPDH has been used to demonstrate equal protein loading. (D) Immunoblot-based verification of altered abundance addressed via immunohistochemistry for the panel of paradigmatic proteins shown in Figure 5 utilizing spleen of two idependent wildtype and woozy animals, respectively. Investigation confirmed a decrease for SIL1, GRP94, PHGDH, ACTA, & CALM as well as an increase for RAB11-FIP1,α-Syn & SELH. GAPDH has been used to demonstrate equal protein loading.