Clean blot ip detection reagent

Immunoprecipitation assays were carried out following a modified version of the protocol provided by Millipore, USA. Treated samples were washed in ice cold PBS and gently lysed in RIPA buffer. The cell lysates obtained were subjected to pre-clearing with BSA-blocked Protein A beads (Bangalore Genei, India) for 30 min at 4°C and slow rotation. The amount of protein in the supernatant was quantified and equal amount of protein was used for pull down from each treatment condition; using Protein A beads pre-conjugated with the antibody of interest or isotype control IgG antibody. After incubation of the whole cell lysates with the antibody-complexed beads for 4 h at 4°C on slow rotation, the pellet containing the bead-bound immune complexes were washed with RIPA buffer twice. The complexes were eluted by boiling the beads in Laemmli buffer for 10 min. The bead free samples were resolved by SDS-PAGE and the target interacting partners were identified by immunoblotting. Clean-Blot^™ IP Detection Reagent (21230) was obtained from Thermo Scientific.

Anti-SNAP-25, anti-actin, anti-Flag, anti-GST, anti-syntaxin, peroxidase conjugated anti-rabbit IgG and anti-chicken IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-SNAP-25^pThr138 (Abgent, San Diego, CA, USA), anti-MYPT1^1-296 [20 (link)], anti-PP1cδ (Millipore, Billerica, MA, USA), anti-GAPDH (Santa Cruz, CA, USA), anti-CPI17^pThr38 [22 (link)], horseradish peroxidase-linked anti-mouse IgG (Cell Signalling, Danvers, MA, USA), Clean-Blot IP Detection Reagent (Thermo Scientific, Waltham, MA, USA) and Texas Red-X phalloidin (Life Technologies, Carlsbad, CA, USA) were purchased as indicated. Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 546-conjugated anti-mouse IgG, Alexa Fluor 546-conjugated anti-goat IgG and To-Pro-3 iodide were obtained from Molecular Probes (Eugene, OR, USA).

Immunoprecipitation assay was performed as per the standard protocols. For immunoprecipitation, 0.5–1 mg of protein from lysate was incubated with 2 µg of appropriate antibody or control IgG antibody overnight. Protein A/G-conjugated agarose beads were used to capture the immune complexes. After a brief centrifugation, supernatant was discarded, and beads were washed thrice with ice-cold PBS (1X) 1000 rpm at 4°C for 1 min. The immunoprecipitated protein was resolved by SDS-PAGE after boiling the beads at 95°C for 5 min in 2X laemmli buffer (Bio-rad) and transferred to a PVDF membrane (GE, Cat#10600023). The membrane was blocked by a solution of 5% non-fat dried milk in TBST buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 hr at RT. Membrane was washed thrice with TBST buffer and probed with primary antibody at 4° for overnight. After washing thrice with TBST buffer and the membrane was probed with HRP-conjugated secondary antibody (Clean-Blot IP Detection Reagent, Thermo Scientific or Mouse monoclonal SB62a Anti-Rabbit IgG light chain (HRP) (ab99697) prepared in 1% milk at RT for 1 hr. Blot was washed thrice with TBST (1X) and chemiluminescent signals were captured using Clarity ECL Western Blotting Substrate (BioRad) in a chemiluminescence imager (Chemidoc Touch, Biorad).

Testes dissected from sv/+ males were homogenized with a Dounce tissue grinder in ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM MgCl₂, 1% NP-40, 5 mM ATP) supplemented with 1 × cOmplete Protease Inhibitor Cocktail (Roche) and centrifuged at 15,000 × g for 10 min at 4 °C. The lysates were pre-cleared with Protein A-Sepharose CL-4B (GE Healthcare) for 1 h at 4 °C and spun briefly, and then supernatants were transferred to fresh tubes. Next, samples were incubated with 5 μg of anti-MYO6 antibody for 1 h at 4 °C with end-over-end mixing, before incubation with Protein A-Sepharose for 1 h at 4 °C followed by four washes with ice-cold lysis buffer. Co-immunoprecipitated proteins were eluted from the beads using 4 × SDS sample buffer and analyzed by SDS-PAGE followed by western blotting. The primary antibodies were detected using Clean-Blot IP Detection Reagent (Thermo Scientific). Co-immunoprecipitation experiments were repeated three times.

To obtain independent confirmation of Numb association with Septin 7, Numb IP was performed with anti-Numb antibody as described above. For this analysis we used three independently obtained samples of C2C12 myotubes different from those used for LC/MS/MS. The immunoprecipitated material was analyzed by Western blot using anti-rabbit polyclonal anti-Septin 7 (Proteintech, 13818-1-AP, 1:1000 dilution). To confirm Numb IP the blot was then probed with anti-Numb antibody (Cell Signaling #2756). Secondary detection was performed with Clean-Blot IP detection reagent (ThermoFisher, 21230, 1:1500).

Cells harvested from the appropriate culture were lysed and the soluble fraction of the cell extract was subjected to western blot and immunoprecipitation were conducted as previously described [11 (link), 24 (link)]. Clean Blot IP Detection Reagent was purchased from Thermo Scientific.