Rabbit polyclonal anti ha

Protein extracts were prepared, and proteins were separated by SDS-PAGE and analyzed by a Western blot as formerly described [4 (link)]. Protein extracts from HA-tagged Orm2 cells were obtained by using a NaOH-based protocol as previously described [52 (link)]. For the phosphate-affinity electrophoresis, protein samples were resolved in the SDS-PAGE gels that contained 6% acrylamide, 10 μM Phos-tag™ affinity reagent (Wako Chemicals USA, Inc.) and 20 μM Mn²⁺. The proteins tagged with 3xHA and 13xMyc (Pah1-Myc) were visualized by using an anti-HA rabbit polyclonal (1:2,000; cat# sc-805; Santa Cruz Biotechnology, Santa Cruz, CA) and an anti-Myc (1:2000; cat# sc-40, Santa Cruz Biotechnology) antibody, respectively. Mouse monoclonal Glucose 6 Phosphate Dehydrogenase (G6Pdh) antibody (1:3,000; cat# sc-47724) and rabbit antiPma1 (1:10,000; kindly provided by R. Serrano), were used as loading control of cytosolic and membrane-associated proteins, respectively. The secondary antibody used was horseradish peroxidase-conjugated goat anti-rabbit (1:2,000; cat# 7074; Cell Signaling, Danvers, MA, USA) or rabbit anti-mouse (1:5,000, cat# P0260; Dako, Carpinteria, CA). Blots were done and images were captured as described elsewhere [4 (link)].

Nuclear and cytosolic fractions of hA treated or non-treated (control) RIN-m5F cells were prepared using cell fractionation kit (Cell Signaling). The samples were precleared with magnetic Dynabeads (Invitrogen), at 4°C, under rotation, for 2 h. Ten micrograms of anti-hA rabbit polyclonal (Santa Cruz) was coincubated with 50 μl proteinG Dynabeads (Invitrogen) at 4°C, under rotation, for 24 h. The Dynabeads–IgG complex was then cross-linked with 1 ml 20 mM DMP (dimethylpimelimidate dihydrochloride) solution in 0.2 M triethanolamine pH 8.2 and reaction terminated with Tris/HCl pH 8 buffer after 30 min. The precleared nuclear and cytosolic fractions and cross-linked Dynabeads were then co-incubated at 4°C, under rotation, for 24 h. After three washes in PBS, the immunoprecipitated material was detached from the magnetic beads with 5× Laemmli buffer and resolved by SDS/PAGE on 4–15%Tris/glycine gels. Western blot analysis using anti-20sα4 or anti-19S Rpn8 primary antibody (1:200) was then performed. Blots were washed three times with PBS-Tween buffer, incubated with goat anti rabbit HRP linked secondary antibodies (1:2000) for 1 h and developed using a chemiluminescence method.