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. 2004 Aug;78(15):7945–7957. doi: 10.1128/JVI.78.15.7945-7957.2004

FIG. 1.

FIG. 1.

Generation and characterization of anti-140K antibodies. (A) Schematic representation of the genomic organization of TYMV RNA. Open bars denote viral ORFs. The encoded 206K protein is proteolytically processed at a peptide bond signified by a filled diamond. Protein domains within the 140K protein are indicated (MT, methyltransferase; PRR, proline-rich region; PRO, proteinase; HEL, helicase), and the locations of peptides used for immunization are indicated by filled squares. (B) 35S-labeled proteins obtained through in vitro translation of TYMV RNA (lane 1) were immunoprecipitated with anti-140K antiserum (lane 2). The proteins were separated by SDS-PAGE on a 10% polyacrylamide gel and were revealed by autoradiography. The major labeled products synthesized by TYMV RNA (206K, 140K, 69K, and 66K proteins) are indicated on the right. (C) Total proteins from healthy (H) and TYMV-infected (I) Arabidopsis leaves (lanes 1 to 4) and healthy and TYMV-infected Chinese (Ch.) cabbage leaves (lanes 5 to 8) were analyzed by SDS-PAGE on an 8% polyacrylamide gel. The proteins were extracted by a standard method (47) (lanes 1, 2, 5, and 6) or under conditions optimized for the extraction of membrane proteins (23) (lanes 3, 4, 7, and 8). 35S-labeled proteins obtained through in vitro (i.v.) translation of TYMV RNA were loaded in parallel lanes of the same gel (lanes 9 and 9′). The proteins were electroblotted onto a nitrocellulose filter and were revealed both by Western blotting with anti-140K antiserum (lanes 1 to 9) and by autoradiography (lane 9′). The positions of molecular weight markers (Biolabs) are indicated on the left (in thousands). The position of the 140K protein detected in infected plants is indicated by asterisks in lanes 4 and 8. (D) Arabidopsis protoplasts were transfected with TYMV RNA (lane 1), water (lane 2), or expression plasmid pΩ-140K (lane 3) or pΩ-206K (lane 4). The cells were harvested 24 h (lane 1) or 48 h (lanes 2 to 4) posttransfection. Total proteins were subjected to SDS-8% PAGE and immunoblot analysis with anti-140K polyclonal antibodies. The positions of molecular weight markers (Biolabs) are indicated on the left (in thousands).