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. 2006 Mar 15;39(2):93–103. doi: 10.1111/j.1365-2184.2006.00373.x

Figure 5.

Figure 5

Immune precipitation of STAT‐1α involved in the JAK‐STAT pathway in KMB‐17 cells treated with IFN‐α and bound by specific antibody to TIIMPSC. (a) Immunoprecipitation of STAT‐1α in KMB‐17 cells stimulated by TIIMPSC antiserum. IFN‐α‐treated KMB‐17 cells were interacted with TIIMPSC antiserum in 32P‐phosphate labelling media, and were lysed in RIPA. The extract of cells was immunoprecipitated with STAT‐1α antibody. The conjugated complex was then absorbed to protein A–Sepharose 4B beads. After washing with RIPA, the samples were eluted in sample buffer and were run on a 12% SDS–PAGE gel. Lane 1, extract of negative cells control, which were not treated with TIIMPSC antiserum, immunoprecipitated with STAT‐1α antibody; lane 2, extract of cells treated with TIIMPSC antiserum, immunoprecipitated by STAT‐1α antibody. (b) Identification of the STAT‐1α phosphorylated form in cells stimulated with TIIMPSC antiserum. IFN‐α‐treated KMB‐17 cells interacted with TIIMPSC antiserum in 32P‐phosphate labelling media and lysed in RIPA. This extract of cells was immunoprecipitated with STAT‐1α antibody and phosporylated tyrosine antibody. The conjugated complex was then absorbed to protein A–Sepharose 4B beads. After washing with RIPA, the samples were eluted in sample buffer and run on a 12% SDS–PAGE gel. Lane 1, extract of cells treated by TIIMPSC antiserum immunoprecipitated with STAT‐1α antibody; lane 2, extract of cells treated with TIIMPSC antiserum immunoprecipitated with phosporylated tyrosine antibody. (c) Immunoprecipitation of Erk in KMB‐17 cells stimulated with TIIMPSC antiserum. IFN‐α‐treated KMB‐17 cells were interacted with TIIMPSC antiserum in 32P‐phosphate labelling media and were lysed in RIPA. The extract of cells was immunoprecipitated with Erk antibody. The conjugated complex was then absorbed to protein A–Sepharose 4B beads. After washing with RIPA, the samples were eluted in sample buffer and run on a 12% SDS–PAGE gel. Lane 1, extract of negative cells control, which were not treated with TIIMPSC antiserum, immunoprecipitated with Erk antibody; lane 2, extract of cells treated with TIIMPSC antiserum, immunoprecipitated with Erk antibody.