Anti ha ab9110

Primary antibodies used in this work were rabbit polyclonal anti-HA (ab9110) from Abcam (Cambridge, UK); mouse monoclonal anti-RAB7A (sc376362), anti-vimentin (sc6260), anti-p-αPAK (sc-135755), anti-αPAK (sc-166887), anti-cofilin-1 (sc-53934), anti-caspase 9 (sc-56073), rabbit polyclonal anti-myc (sc-789), anti-p-AKT 1/2/3 (sc7985-R), anti-AKT 1/2/3 (sc8312), anti-p-PKA α/β/γ cat (sc32968), anti-PKA (sc903), and ROCK-2 (sc5561), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal anti-β-catenin (610154) from BD Transduction Laboratories (San Jose, CA, USA); rabbit polyclonal anti-cleaved caspase 9 (#7237), anti-NF-kB p65 (#8242), anti-matrix metalloproteinase 2 (MMP2) (#4022), anti-p-vimentin S38 (#13614), and anti-p-p65/RelA (Ser536) (#3033) from Cell Signaling Technology (Leiden, The Netherlands); mouse monoclonal anti-tubulin (T5168) from Sigma-Aldrich and rabbit polyclonal p-ROCK2 (GTX122651) from GeneTex (Irvine, CA, USA). Secondary antibodies conjugated to fluorochromes (for immunofluorescence analyses) or horseradish peroxidase (HRP, for immunoblot analyses) were from Invitrogen (Carlsbad, CA, USA) or Santa Cruz Biotechnology (Santa Cruz, CA, USA).

pCCA1:CCA1-HA-YFP/cca1-1 and 35S:AFR-MYC transgenic plants were used for ChIP. Anti-MYC (06-599, Millipore), anti-HA (ab9110, Abcam), and anti-H3ac (05-724, Millipore) antibodies and salmon sperm DNA/protein A agarose beads (Millipore, Billerica, MA, United States) were used for chromatin immunoprecipitation. DNA was purified using phenol/chloroform/isoamyl alcohol and sodium acetate (pH 5.2). The level of eluted DNA fragments was quantified by quantitative real-time PCR using specific primer sets (Supplementary Table S2). The values were normalized to the input DNA level.

Lysates used for immunoblotting were prepared in ice-cold RIPA Buffer containing HALT protease inhibitors (ThermoFisher). Clarified cell lysates were resolved on 4–15% mini-PROTEAN TGX pre-cast gels (Bio-Rad), and transferred to nitrocellulose membranes using a TransBlot Turbo (Bio-Rad, Hercules, CA, USA). Membranes were blocked with Odyssey TBS Blocking Buffer (Li-Cor, Licoln, NE, USA) and then probed with the appropriate antibodies. Primary antibodies were diluted in TBS + 1% PVP as follows: anti-PDX1, ab47267 1:5000 (Abcam, Cambridge, MA, USA); anti-PDX1, sc-14664, 1:5000 (Santa Cruz, Dallas, TX, USA) anti-Tubulin, T5326, 1:20000 (Sigma); anti-βactin, A5316, 1:5000 (Sigma); anti-eGFP, ab290, 1:5000 (Abcam); anti-HA, ab9110, 1:5000 (Abcam); anti-mCherry, ab125096, 1:2000 (Abcam) anti-OLLAS; NBP1-06713, 1:1000 (Novus, New York, NY); anti-FLAG, F3165-.2MG, 1:400 (Sigma); anti-His, 12698, 1:1000 (CST, Danvers, MA); anti-myc, 2278, 1:1000 (CST), anti-V5, MA5-15253, 1:1000 (ThermoFisher). Membranes were incubated with appropriate antibodies overnight at 4°C. Secondary antibodies (Li-Cor) were diluted 1:10 000 in Odyssey TBS Blocking Buffer (Li-Cor) with 0.2% Tween 20 added and incubated with membranes for 1 h at room temperature. Immunoblots were developed using a Li-Cor Odyssey CLx.