β tubulin antibody

The cells were lysed with RIPA buffer (25 mM Tris-HCl pH7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). The concentration of the protein lysate was quantified by Bradford assay, using Bradford reagent (Bio-Rad, USA). Equal amounts of protein samples (50 μg) were separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked with 5% bovine serum albumin (BSA, Sigma, USA) for 1 h at room temperature and then incubated overnight at 4°C with 1 : 1000 dilutions of the appropriate primary antibodies (p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2) from Cell Signaling Technology (USA) and 1 : 500 dilution of β-tubulin antibody from Santa Cruz Biotechnology (USA), followed by incubation with 1 : 10000 dilution of the secondary Ab at room temperature. After each incubation, the membranes were washed with TBS-Tween 20. To visualize the signals, the membranes were incubated with enhanced chemiluminescence (ECL) solution and developed using ChemiDoc Imaging System (Invitrogen, iBright CL1000, USA).

BxPC-3 and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 and DMEM, respectively. The media were supplemented with 10% FBS and 1% Pen/Strep. Anti-EphA2 antibody (1C11A12) and HRP-conjugated goat anti-mouse secondary antibody were purchased from Thermo Fisher Scientific and β-tubulin antibody was purchased from Santa Cruz Biotechnology.

Cells were lysed with RIPA buffer containing 1% Triton X-100, protease inhibitors and phosphatase inhibitors. Total protein lysates were quantified, and lysates containing 50 μg of total protein were separated by SDS-PAGE and then transferred onto nitrocellulose membranes and subjected to Western blot analysis as previously described [28 (link)]. Densitometry of Western blots was quantified using Quantity One 1-D analysis software (Bio-Rad Laboratories, Hercules, CA, USA).
To obtain nuclear extracts, cells were lysed with phosphate-buffered saline (PBS) containing 1% Nonidet P-40. The nucleus were washed in the lysis buffer multiple times and lysed with loading dye containing SDS, as described in a protocol developed by others [29 (link)].
The primary antibodies used for Western blot analysis were rabbit polyclonal Smad2/3 antibody (sc-8332; Santa Cruz Biotechnology), rabbit phospho-Smad3 antibody (9520; Cell Signaling Technology, Danvers, MA, USA), goat polyclonal BCAR3 antibody (sc-47811; Santa Cruz Biotechnology), rabbit polyclonal p130Cas antibody (sc-860; Santa Cruz Biotechnology), rabbit polyclonal USF-2 antibody (sc-862; Santa Cruz Biotechnology) and mouse monoclonal β-tubulin antibody (sc-5274; Santa Cruz Biotechnology). All corresponding secondary antibodies were purchased from Santa Cruz Biotechnology.

To analyse JAZ1 protein stability during ETI, 10 mM MgSO₄, Psm ES4326/avrRpt2 or Psm ES4326/avrRpt2 with 40 μM MG115 was infiltrated into the 3rd and 4th leaves of 3-week-old plants at OD_600nm=0.2. After 4 h, 0.2 g of the infiltrated leaves were collected for each sample. To measure the JAZ1 stability affected by SA in soil growing plants, 1 mM SA were sprayed on 3-week-old plants for 4 h. For the time course assay of JAZ1 stability, 2-week-old seedlings on MS plates were sprayed with 200 μg ml⁻¹ CHX (Mock), 1 mM SA plus 200 μg ml⁻¹ CHX (SA) or 100 μM MeJA plus 200 μg ml⁻¹ CHX (MeJA), and samples were collected at the corresponding times. Total protein was extracted using the extraction buffer mentioned above and denatured by heating at 95 °C for 5 min after adding the protein loading buffer containing 100 mM dithiothreitol. The HA-JAZ1 protein level was detected by western blotting using the HA antibody. β-Tubulin was also detected as an internal control using the β-Tubulin antibody (Cat No. sc-166729, Santa Cruz Biotechnology; dilution, 1:2,000)58 (link). Full versions of cropped blots are shown in Supplementary Fig. 19.

Cell lysates were mixed 1:1 with 2 × SDS-PAGE sample buffer and incubated for 5 min at 95 °C. Afterwards, they were separated by SDS-PAGE electrophoresis and subsequently electrotransferred onto nitrocelulose membrane (Amersham). Membranes were blocked with 5% BSA (Bioshop) in Tris-Buffered Saline with Tween 20 (TBST). Membranes were incubated with primary antibodies (Caveolin-1 N-20 Antibody (sc-894, Santa Cruz Biotechnology) diluted 1:1000 in 2.5% BSA in TBST and β-tubulin Antibody (sc-134234, Santa Cruz Biotechnology) diluted 1:2000 in 2.5% BSA in TBST for 2 h and washed in TBST. Subsequently, membrane was incubated with HRP-labeled anti-rabbit IgG antibody (A0545, Sigma) diluted 1:20 000 in 1% BSA in TBST) for 1 h and finally the signal was developed using the ECL system (Amersham).