Fig 1: Development and validation of the ?-secretase epsilon-cleavage assay to quantitatively determine relative C99 cleavage by ?-secretase in cells. (a) Schematic overview of the ?-secretase epsilon-cleavage assay. Upon membrane cleavage of the C99 hybrid protein by ?-secretase, Aß peptides are released into the medium and the AICD-T4L-rTA hybrid protein is released from the membrane into the cytoplasm. This allows the hybrid protein to enter the nucleus and to bind tetO DNA-binding site to stimulate luciferase reporter gene activity as measurement for total C99 cleavage, both by endogenous and by transfected ?-secretase variants. (b) Relative reporter gene activity using C99-T4L-rTA. Rho(4M)-TEV-site-rTA and Arr(3A)-TEV serve as positive control using a conventional Tango protein–protein interaction assay. (c) Cleavage of the C99 hybrid substrate is affected by ?-secretase inhibitors (100 nm per well), indicating that cleavage in HTL cells is due to endogenous ?-secretase. (d) IC50 dose–response curves for the two most potent ?-secretase inhibitors (LY-411575 compound and compound E (Cpd E)). (e) Immunoblot validation of CRISPR/Cas9-mediated chromosomal PS1 and PS2 deletions. PS1 and PS2 protein levels of wildtype (WT) HTL and double-deletion cells are determined by immunoblotting using antibodies that detect the PS1 C-terminal fragment (CTF) (Cell Signaling Technologies 3622S) and antibodies that detect PS2 CFT (Abcam ab106351). ß-actin antibody (Abcam ab6276) is used for normalization. *Antibody cross-reactive band. #PS2 membrane protein oligomers. (f) Chromosomal deletion of PS1 and PS2 abolishes reporter gene activity. Activity can be restored by transfecting WT PS1 gene to a similar level as the positive control of Rho(4M)-TEV-site-rTA and Arrestin(3A)-TEV. (error bars=s.e.m., n=3, P-values (two-tailed Student’s t-test versus control (a, f) or WT (c)): *P<0.05; **P<0.01; ***P<0.001).
Fig 2: Altered expression of membrane proteins in heterozygous SAO red cells. Red cell membranes from SAO and control peripheral blood samples were prepared, separated by SDS-PAGE and immunoblotted. The antibodies directed against protein 4.2, GPA, RhAG, Rh polypeptides, CD47, aquaporin-1, GPC, stomatin and phospho-Tyr359 band 3 were in-house rabbit polyclonal antibodies. Commercial antibodies were used to detect actin (ab6276) and peroxiredoxin-2 (PRDX2; ab109367) both from Abcam, Cambridge, United Kingdom. Monoclonal antibodies were used to detect band 3 (BRIC170), alpha-spectrin (BRIC174), beta-spectrin (BRAC65), GPB (and to a lesser extent GPA, R1.3), ICAM-4 (BS46/BS56), LFA-3 (BRIC5), DAF (BRIC128) and Lutheran (BRIC221). SAO het and SAO het (F): Membranes prepared from the heterozygous father of the homozygous SAO child. SAO het (M): Membranes prepared from the heterozygous mother of the homozygous SAO child. LC, Loading control.
Fig 3: Expression of membrane proteins involved in reticulocyte maturation. Red cell membranes from heterozygous SAO and control peripheral blood samples were prepared, separated by SDS-PAGE and immunoblotted. The antibody directed against CD44 was an in-house rabbit polyclonal antibody. Commercial antibodies were used to detect actin (ab6276), calreticulin (ab2907); CD147 (ab108308), LAMP-2 (ab25631) and transferrin receptor (TfnR) (ab84036); all from Abcam, Cambridge, United Kingdom. An in-house monoclonal antibody was used to detect beta-spectrin (BRAC65). SAO het: Membranes prepared from the heterozygous father of the homozygous SAO child. LC: Loading control.
Fig 4: GCL and GAGE proteins are co-expressed in human cancer cells lines.GCL and GAGE protein expression was examined in 9 human cancer cells lines derived from cervix (HeLa), colon (HCT116), melanoma (MZ2-MEL), breast (BrCa-MZ01, SK-BR3, T47D, M4A4, MCF7) and embryonic cancer (NTERA2) using Western blotting. A375 melanoma cells with exogenous expression of GCL were included as positive control. Antibodies: GCL pAb1, Sigma Aldrich; GCL pAb2, clone A14, Santa Cruz Biotech; GAGE mAb, clone M3 [4], beta-actin mAb, Ab6276; Abcam.
Fig 5: Effects of EYA1 knockdown on expression of cyclin D1 and phosphorylated ?H2AX histoneImmunoblots were performed using specific antibodies against cyclin D1 and phosphorylated ?H2AX proteins using cell lysates prepared from stable A375 melanoma cells containing ShRNA vectors (control vector, and vectors with ShRNA1 or ShRNA2 of EYA1 gene), as described in the text. Panel A: Representative immunoblots using antibodies against EYA1 (Proteintech 22658-1-AP, 1:600), Cyclin D1 (Santa cruz, sc-20044, 1:100), ?-H2AX (Abcam, ab11174 1:1000), and ß-Actin (Abcam, [AC-15] ab6276, 1:1000); Panel B: Expression levels were quantified using ImageJ software and normalized to ß-Actin. Plotted are averages of three independent experiments. * p<0.05.
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