Wild-type and apn1 mutant embryos and dissected larval tracheae were homogenized on ice using a Dounce tissue grinder in 1mL of lysis buffer containing 130mM NaCl, 50mM Tris-HCl pH = 8, 0,5% Triton-X and protease inhibitor (Roche). After 30min at 4°C under rotation the homogenate was centrifuged for 20min at 14.000rpm. Sample buffer 3x SDS was added to the supernatant and boiled for 5min at 95°C.
Proteins were separated by SDS-PAGE and blotted onto nitrocellulose 0.45 membrane (Amersham). After blocking in 5% BSA+TBST, the membrane was incubated overnight with rabbit anti-Apn diluted 1:1000, rat anti-Crb[88 (link)] diluted 1:1000 and mouse anti-alpha-tubulin (Sigma) diluted 1:5000 in blocking buffer. Peroxidase antibodies were used for detection.
Proteins were separated by SDS-PAGE and blotted onto nitrocellulose 0.45 membrane (Amersham). After blocking in 5% BSA+TBST, the membrane was incubated overnight with rabbit anti-Apn diluted 1:1000, rat anti-Crb[88 (link)] diluted 1:1000 and mouse anti-alpha-tubulin (Sigma) diluted 1:5000 in blocking buffer. Peroxidase antibodies were used for detection.