Figure 12.
Immunopurification of phot1-GFP from Arabidopsis.
(A) Identification of putative phot1-interacting proteins by LC-MS/MS of phot1-GFP immunoprecipitations. ADL, Arabidopsis dynamin-like; TUA, tubulin α-chain; TUB, tubulin β-chain; AHA2, Arabidopsis H+-ATPase 2; PIP3, plasma membrane intrinsic protein 3; HSC70-1, heat shock cognate 70 kD protein 1; and RPT2, root phototropism 2. TAIR accession numbers are given where MS data match a single accession.
(B) phot1-GFP and phot2-GFP associates with clathrin in vivo. Four-day-old light grown seedlings were dark adapted overnight and treated with 100 μmol m−2 s−1 of blue light for 30 min. phot1-GFP and phot2-GFP were immunoprecipitated from the microsomal membrane fraction with anti-GFP antibody linked to magnetic microbeads and subjected to immunoblot analysis with anti-GFP and anti-clathrin antibodies. The plasma membrane marker line, GFP-Lti6b, was used as a control for nonspecific interactions. Sizes of molecular weight marker proteins are indicated on the left in kilodaltons.