Protein assay kit

To investigate the phosphorylation levels of Hog1 and Slt2, yeast cells expressing Hog1-HA and Slt2-HA fusion proteins were first grown overnight in SC medium at 30 °C. Cells were then cultured in fresh SC medium to an OD₆₀₀ of 0.8–1.0, and treated with 50 µM CdCl₂ or 1 µg/mL TM for an additional 2 h. To prepare protein extracts, cell cultures were first cooled in ice water for 10 min and cells were harvested by centrifugation at 4 °C, then washed once with ice-cold water. Next, cells were resuspended in ice-cold lysis buffer [23 (link)] and vortexed with glass beads 10 times for 1 min each time. The lytic samples were centrifuged at 12,000 rpm for 10 min at 4 °C. Protein concentration was measured using a protein assay kit (Sangon Biotech, Shanghai, China) before samples were boiled with sample buffer (250 mM Tris-HCl pH 6.8, 10% sodium dodecyl sulphate (SDS), 0.05% Bromophenol Blue, 50% glycerol, 7.5% DTT) for 5 min.
To perform the Western blotting assay, samples were first analysed by 10% SDS-PAGE and transformed onto nitrocellulose membranes. Phosphorylation of Hog1-HA or Slt2-HA protein was immunodetected by phospho-p38 MAPK or phospho-p44/42 MAPK antibody (New England Biolabs, Beverly, MA, USA), respectively. Expression of Hog1-HA and Slt2-HA fusion proteins was detected by HA monoclonal antibody (Sangon Biotech).

The partial cDNA sequence of AjCTRP9 was cloned with specific primers (Table S1 in Supplementary Material) then double digested with EcoRI and NotI and ligated into the vector pGEX-4T-2 (GE, USA). Ajβ-actin was used as a control, as described in our previous work (38 (link)). The recombinant plasmid (pGEX-4T-2-AjCTRP9) was transformed into Escherichia coli cells of Transetta (DE3) (TransGen, China), and the DNA was sequenced to ensure correct orientation. The positive transformants were subsequently incubated in LB medium containing 50 μg mL⁻¹ of ampicillin at 37°C and 200 rpm. Then, the expression of the GST-tagged fusion protein was induced in pGEX-4T-2-AjCTRP9 with 1 mM isopropyl-b-d-thiogalactopyranoside (IPTG) at 37°C for 4 h, and the protein was collected after cells were disrupted with ultrasonic waves. The GST-Sefinose™ Resin was used for the purification, according to the manufacturer’s protocol (Sangon, China). The concentration of the purified soluble protein was quantified with a Protein Assay Kit (Sangon, China). The soluble target protein, after dialysis, was injected into a 4-week-old mouse to acquire polyclonal antibody according to a previously described protocol (38 (link)).