Fig 1.
Two monoclonal antibodies recognize CAM-cys modified peptide sequences.
(A) MYADML2 peptides used to screen for reactivity to modified cysteines lack the first anchoring cysteine present in the immunogen. Cysteine residues are highlighted in blue in peptide #1, which contains only natural amino acids. Peptide #20 was synthesized with two CAM-cys residues shown in red. An aliquot of peptide #1 was reacted with IAM (5 mM) for two hours at 37°C prior to application to membranes in the middle row of (B-D). One microliter dots of these peptides were applied to nitrocellulose at decreasing concentrations (1,000, 100, 10, and 1.0 μg/mL; denoted by triangles in B-D). Finally, membranes were probed with monoclonal antibodies 4E7 (B) and 52H11 (C) followed by secondary antibodies or with secondary antibodies alone (D) as a control.
Fig 2.
Two monoclonal antibodies recognize multiple proteins in cell lysates treated with IAM.
Whole cell lysates from 293 cells were treated with increasing concentrations (0, 0.1, 0.5, 2, 5mM) of cysteine alkylating compounds IAM (A, D, G), acrylamide (B, E, H), and NEM (C, F, I). After Western blotting, panels of proteins were probed with 4E7 (A, B, C), 52H11 (D, E, F) or the NEM-cys targeted antibody OX133 (G, H, I). Lanes marked by 0 contain unmodified protein that serves as a baseline control. Labels in black triangles symbolize treatment dose dependent in IAM (I), acrylamide (A), and NEM (N). In B, C, E, F, and I, lane labeled I on the far right indicates lysates treated with IAM alone (2 mM). In G and H, the lane on the far right labeled with N contains lysates treated with NEM alone (2 mM). TUB = western blot for tubulin as a loading control.
Fig 3.
Effect of competitive blocking by cysteine alkylation on antibody binding to IAM-treated proteins.
Potentially competitive cysteine alkylating agents were added either before or after IAM to determine whether cysteine modification could affect recognition of proteins incubated with IAM. Each sample of whole 293 cell lysate was sequentially exposed to different combinations of alkylators (labeled “First” then “Second”). The first lane of each panel was not treated with alkylators. The second lane was treated with IAM only. The third lane was treated first with NEM and then with IAM. The fourth lane was treated with acrylamide and then with IAM. The fifth lane was treated with IAM then NEM. And the sixth lane was treated with IAM followed by acrylamide. I = IAM (2mM); A = acrylamide (2mM); N = NEM (2mM). After Western blotting, membranes were probed with 4E7 (A) or 52H11 (B).
Fig 4.
Detection of IAM labeled proteins from living cells.
IAM was added to the growth media of 293 cells which were labeled at 37°C before fixation with formalin and staining with 4E7 (A-D) or 52H11 (E-H), all at 1:50 dilutions. The following IAM concentrations were applied for 10 minutes: None (A, E), 100μM (B, F), and 2mM (C, G). In some wells (D, H), NEM (100μM) was added to the media for 5 minutes before addition of IAM for 5 minutes.
Fig 5.
Detection of protein CAM-cys after pulse chase labeling of living cells with IAM.
IAM (1mM) was added to growth media of 293 cells for 2 minutes followed by media without IAM for defined periods of time. Chase time intervals (lanes marked by triangle t) were 0, 8, 24, and 32 hours. Cells were collected for analysis of labeled protein content by Western blotting with 4E7 (A) and 52H11 (B). Western blotting for tubulin (TUB) served as a control for total protein loading.
Fig 6.
Antibodies to CAM-cys selectively target purified proteins.
A series of purified proteins were treated in the presence or absence of the non-thiol reductant TCEP (2.5mM) with IAM or NEM (5mM). Proteins were then analyzed by Western blotting with 4E7, 52H11, or OX133. Selected CAM-cys labeled proteins reacted with 4E7 and 52H11. Equal loading of proteins was demonstrated by staining membranes with a non-selective protein stain (Revert, LI-COR). Proteins migrated at the expected molecular masses as follows (approximations): beta casein (27 kDa), BSA (65 kDa), BGN (40 kDa), DCN (38 kDa), COL1 (>250 kDa), COL4 (>250 kDa), JAG1 (170 kDa), IL17RC (75 kDa), LAMA2 (>180 kDa), vWF (260 kDa), TSP1 (150 kDa), TSP2 (155 kDa).
Fig 7.
Peptide sequences targeted by 4E7 and 52H11.
(A) Schematic of sequences fused to GFP in cDNA expression constructs. Wild type human MYADML2 fragment (W) and three variants with mutations in cysteines (to serine marked in red; C1, C2, and Cb) were transfected into cells. Lysates from transfectants were incubated at 37°C for 30 min with or without IAM (5 mM) were analyzed by GFP (B), 4E7 (C) and 52H11 (D) Western blotting. + indicates the molecular weight of peptide-fused GFP proteins. (E) a series of mutant peptides corresponding to the immunization epitope were analyzed by dot blotting. For IAM treated dots, peptides were incubated with IAM 5mM at 37°C for 30 minutes. Each dot contains 1 μg of each of the peptides shown, spotted on nitrocellulose.
Fig 8.
Sequences that do not react with CAM-cys antibodies.
All cysteine residues from proteins that were found to be unreactive to both 4E7 and 52H11 after IAM treatment were analysed as follows: The four residues surrounding each side of the cysteines were arrayed for each of the eight positions relative to the cysteine. The relative frequency of each amino acid was then determined (percentage of all residues) and displayed by amino acid by position in a heat map. The most common amino acids at each position are listed at the top of the map.