Figure 3.

Identification of p97 tyrosine phosphatase from low-density microsomes. (A) Cytosol or LDM were incubated as described above in the presence or in the absence of 100 μM bpV(phen) and 100 μCi [γ-³²P]ATP. Lysates were then incubated in the presence of glutathione-Sepharose beads carrying 15 μg of GST-PTPH1 D811A mutant for 4 h at 4°C. Proteins pulled-down were resolved by SDS/PAGE and either immunoblotted with anti-p97 or anti-GST antibodies and revealed by enhanced chemiluminescence (Middle and Bottom) or directly radioautographed to X-Omat AR films (Top). A representative experiment is shown (n = 3). (B) Analytical fractionation by isopycnic density gradient centrifugation of LDM. Each fraction was tested for its content p97, PTPH1, JAK-2, and Bip by immunoblotting. Quantitation was done as described under Experimental Procedures.