Anti gfp jl 8

Embryonic extracts for immunoblotting were prepared by resuspending embryos in an equal volume of 2X SDS-PAGE Sample buffer (100 mM Tris–Cl (pH 6.8);4% SDS; 0.2% bromophenol blue; 20% glycerol; 200 mM β-mercaptoethanol) and homogenizing with a pestle in a microfuge tube. To make S2 cell extracts 1 mL of resuspended S2 cells were spun down in a microfuge tube, the media was removed, and the pellet resuspended in an equal volume of 2X SDS PAGE Sample buffer. Samples were boiled for 5 min, spun to clear debris, and 10 μl of the resulting extract run on a 7.5% SDS-PAGE gel, and transferred to a nitrocellulose membrane. To detect the transgenic GFP-tagged Abl proteins we used anti-GFP (JL-8, 1:500 or 1:1000, Clontech). Anti-αTubulin (Sigma, 1:10,000) or anti-Pnut (Developmental studies Hybridoma Bank, 1:30) were used as loading controls. Detection was done using HRP-conjugated anti-mouse IgG secondary antibody (Pierce, 1:50,000), and the ECL plus substrate kit (Pierce).

For protein–protein interactions in gametophores, total protein extracts from gametophores of PpSHR1-3×Myc#16, PpSHR2-3×Myc#46, Citrine–PpSCR1 PpSHR1-3×Myc#29, and Citrine–PpSCR1 PpSHR2-3×Myc#46 plants were prepared with immunoprecipitation buffer (25 mM Tris-HCl [pH 7.5], 75 mM NaCl, 15 mM MgCl₂, 15 mM EGTA, 0.1 mM NP-40, 10 mM NaF, 25 mM β-glycerophosphate, 2 mM sodium o-vanadate, and 1× complete protease inhibitor cocktail [Roche]) and were immunoprecipitated with monoclonal anti-Myc (4A6; Sigma-Aldrich) or anti-GFP (JL-8; Clontech) antibodies. The crude extracts and immunoprecipitates were examined using anti-Myc and anti-GFP antibodies.

Total cell lysates were harvested using RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 0.25% sodium deoxycholate) with protease inhibitor cocktails (Cat#P8340, Sigma-Aldrich, St. Louis, MO, USA). The antibodies used in this study are described below: anti-GFP (JL-8, Clontech, Mountain View, CA, USA), anti-CPAP (ab221134, Abcam, Danvers, MA, USA), anti-STAT3 (9139, Cell signaling, Danvers, MA, USA), anti-phospho-STAT3/Y705 (9145, Cell Signaling), anti-phospho-p65 (3036, Cell Signaling), anti-p65 (8242, Cell Signaling), anti-cleaved caspase-3 (9661, Cell Signaling), anti-GAPDH (sc32233, Santa Cruz, Dallas, TX) and anti-α-tubulin (DM1A, T6299, Sigma-Aldrich).

Rabbit anti-Ror2 ^{61 (link)} and anti-IFT20 ^{31 (link)} antibodies were prepared as described previously. Sheep anti-TGN46 and rabbit anti-Giantin antibodies have been described previously^{62 (link)}. Following antibodies were purchased commercially: mouse anti-GM130 (35, BD), anti-Cortactin (4F11, Millipore), anti-γ-tubulin (GTU-88, Sigma), anti-tyrosinated tubulin (TUB01A2, Sigma), anti-AKAP450 (15, BD), anti-GFP (JL-8, Clontech), anti-acetylated tubulin (6-11B-1, Sigma), anti-Myc (9E10, Santa Cruz), anti-Golgin-97 (CDF4, Thermo), and Alexa Fluor 647-conjugated anti-GM130 (5G8, MBL); rabbit anti-IFT20 (13615-1-AP, Proteintech), anti-Arl13B (ab83879, Abcam), anti-GM130 (PM061, MBL), anti-γ-tubulin (T5192, Sigma), anti-Golgin-97 (D8P2K, Cell Signaling Technology), and HRP-conjugated anti-α-tubulin (PM054-7, MBL).