Irdye 680lt conjugated anti rabbit igg

Western blotting was performed as previously described.6 Briefly 10⁷ Jurkat T cells were lysed in 200 μl Triton X‐100 buffer and 2·5 × 10⁵ to 7·5 × 10⁵ cells were resolved by SDS–PAGE under reducing conditions. Protein bands were detected by the LI‐COR Odyssey Sa system after developing with rabbit anti‐SAP antibody (clone FL‐128 Santa Cruz Biotechnology (Santa Cruz, CA)), mouse anti‐phosphotyrosine (clone PT‐66, Sigma, St Louis, MO), goat anti‐HA (biotinylated, Vector Laboratories) or anti‐β‐actin (clone AC‐15, Santa Cruz Biotechnology) followed by the appropriate secondary antibody or fluorophore‐conjugated streptavidin (IRDye 680LT‐conjugated anti‐rabbit IgG, IRDye 800 CW anti‐mouse IgG, IRDye 680LT‐conjugated anti‐mouse IgG, IRDye 800 CW Streptavidin; all LI‐COR Biosciences, Lincoln, NE). Quantification of protein expression was performed using LI‐COR odyssey sa software version 1.0.

PNA (Arachis hypogaea lectin, Sigma-Aldrich L0881) was labeled with IRDye 800CW (Licor 928–38044). Salivary glands from wandering third instar larva were dissected in PBS and transferred to a 1.5 ml eppendorf tube containing 50 μl of RIPA buffer (Sigma) containing 1× Halt Protease Inhibitor (Thermo Scientific). Protein extracts from 3 glands was loaded in each lane of a NuPAGE 4–12% Bis-Tris gel (Invitrogen). Gels were transferred onto nitrocellulose membranes. For Li-COR western blotting, the membranes were blocked with Odyssey Blocking Buffer (PBS-based) (Li-COR) and incubated with IRDye 800CW-conjugated PNA (1:5000) overnight at 4 °C. For the tubulin control blots, membranes were incubated with the tubulin antibody (1:1000) (Cell Signaling Technology, #2125) overnight at 4 °C, washed with PBS containing 0.1% Tween-20 (PBST) three times, and incubated with IRDye 680LT-conjugated anti-rabbit IgG (1:10,000) (Li-COR, #926–68021) for 1 h at room temperature. Membranes were washed with PBST three times, with PBS twice, and then scanned using a Li-COR Odyssey Infrared Imaging System. Full-length western blots are shown in Supplementary Fig. 5.