Hrp conjugated goat anti rabbit

Total protein was extracted from pulverized tissue using 200–300 μl of Laemmli (35 (link)) Buffer (0.24 M Tris–Cl pH 6.8, 6% sodium dodecyl sulphate, 30% glycerol, 16% β-mercaptoethanol, 0.006% bromophenol blue, 10 M urea) as previously described (29 ). Extract was run on denaturing 10% or 4–12% Bis-Tris NuPAGE gels with MOPS buffer (Life Technologies), then transferred to nitrocellulose membrane using a tank transfer at 300 mA for 1.5 h (for AvrBs2-HA) or 1 h (for ATR1Δ51-FLAG or OsL5). Membranes were stained for total protein with Ponceau S (0.5% Ponceau S, 1% acetic acid) and the large subunit of RuBisCo (approx. 55 kDa) is shown below western blots as a loading control. Membranes were blocked overnight with 5% milk in TBST buffer (20 mM Tris–Cl, 0.5 M NaCl, 0.05% Tween-20, pH 7.5). AvrBs2-HA protein was detected using 1:1000 of rat Anti-HA HRP-conjugated antibody (Roche, 3F10). ATR1Δ51-FLAG was detected using 1:2000 of mouse Anti-FLAG M2 HRP-conjugated antibody (Sigma, A8592). Antibodies for OsL5–6xHis were made by expressing and purifying exotoxinA-OsL5–6xHis, then submitting this protein to a commercial vendor (Josman, LLC) for generation of OsL5–6xHis-specific antibodies in rabbits. OsL5 protein was detected using rabbit anti-OsL5–6xHis 1:40,000 and goat anti-rabbit HRP-conjugated 1:5000 (BioRad, 170–6515).

Proteins were extracted and Western blotting was performed as described in [26 (link)]. The following primary antibody dilutions were prepared in a PBS solution (0.1 M phosphate buffer was used throughout) containing 0.2% I-Block (Applied Biosystems) and 0.05% Tween-20: anti-BRF1/2 (Cell Signalling Technologies), anti-pan ZFP36-family (SB1/30.13) and anti-β-actin (Abcam). The following secondary antibodies were diluted in a PBS solution containing 0.05% Tween-20: goat anti-rabbit-HRP conjugated (Biorad) or donkey anti-rat-HRP conjugated (Abcam).

For standard western blotting, protein extracts were separated by electrophoresis in precast 4%–15% polyacrylamide gels (Biorad, Hercules, CA). To separate phosphorylated forms of Rif1, protein extracts were run on a 0.5% agarose-strengthened 3% polyacrylamide gel containing 20 μM Mn²⁺-Phostag (AAL-107, Wako Chemicals, Japan). Proteins were then transferred to a PVDF membrane using a wet-transfer system. The membrane was blocked in TBST (TBS with 0.1% Tween-20) supplemented with 2.5% BSA and then incubated in the appropriate primary antibody at a dilution of 1:1,000 overnight at 4 °C. The blot was then washed 3 times for 10 min each in TBST and then incubated in the appropriate secondary antibody at a dilution of 1:10,000 for 1 h at room temperature. The blot was then washed 3 times for 10 min each in TBST and then treated with Pierce SuperSignal West Pico ECL and used to expose autoradiography film. The following antibodies were used: rabbit anti-GFP (ab290, Abcam, Cambridge, United Kingdom), rabbit anti-PP1a (2582, Cell Signaling, Danver, MA), and Goat anti-Rabbit HRP conjugated (Biorad).

Cells were lysed and proteins separated by SDS‐PAGE using 4%–12% Bis‐Tris gels, followed by transfer to PVD membranes, blocking, incubation with primary antibodies overnight at 4°C, incubation with HRP‐conjugated secondary antibodies for 1 hr at room temperature, and detection using enhanced chemiluminescence. Primary antibodies were rabbit anti‐ERCC1 (Santa Cruz #sc‐10785) and mouse anti‐β‐actin (Sigma #A2228). Secondary antibodies were HRP‐conjugated goat anti‐rabbit (Bio‐Rad) and goat anti‐mouse (Bio‐Rad) antibodies.

Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with 2-mercaptoethanol (final concentration 6%) and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), then boiled for 5min at 95°C. Equal amounts (10-30 μg) of samples were loaded and proteins were separated by SDS-PAGE using 4-12 % Bis-Tris gels (Bio-Rad, Hercules, CA, USA), followed by transfer to PVD membranes using iBlot Dry Blotting Gel Transfer System (Thermo Fisher Scientific). Membranes were blocked for 1 h in 5% BSA-TBST at room temperature, and then incubated with primary antibodies overnight at 4°C. Blots were washed and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature, and detection was performed using enhanced chemiluminescence. Primary antibodies were against p16 (Abcam, #ab108349, 1:500 dilution) and p21 (Cell Signaling Technology, #2947s, 1:1000 dilution). Secondary antibodies were HRP-conjugated goat anti-rabbit (Bio-Rad) and goat anti-mouse (Bio-Rad) antibodies. An antibody against beta actin (Sigma, #A2228, 1:10,000 dilution) was used for loading control.

The Na₂CO₃, DTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, HCl, SDS, PMSF, Tris-HCl and proteinase K were all obtained from Sigma-Aldrich. All primary antibodies were obtained from Cell Signaling Technology (Bervely, MA, USA) except for β-actin (Sigma-Aldrich). Secondary antibody, HRP-conjugated goat anti-rabbit were purchase from Bio-Rad Laboratories. Annexin V/PI apoptosis kit was purchased from Invitrogen. MEK ½ inhibitor, U0126, was obtained from Cell Signaling Technology and PI3K inhibitor, Wortmannin, was obtained from Sigma-Aldrich.