Phosphatase inhibitor complex 3

Cells were harvested and resuspended in RIPA cell lysis buffer [200 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100] with 1% PMSF (Biosharp, Hefei, China) and phosphatase inhibitor complex III (1 mM) (Sangon Biotech, Shanghai, China). Samples were separated by 10% SDS-PAGE and then transferred from the gels to polyvinylidene difluoride (PVDF) membranes. After blocked in 5% nonfat milk, the membranes were incubated with the indicated primary and secondary antibodies. Protein bands were visualized with ECL system (Biorad, Berkeley, CA, USA).

Cells were lysed in radio‐immunoprecipitation assay buffer with 1% phenylmethanesulfonyl fluoride (PMSF) (Biosharp) and phosphatase inhibitor complex III (1 mmol/L) (Sangon Biotech, Shanghai, China). Equal amounts of protein extracts were separated by 8% sodium dodecyl SDS‐PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Merck, Darmstadt, Germany). The membranes were blocked in 5% non‐fat milk for 2 hours at room temperature and incubated with the indicated primary antibodies at 4°C overnight. After being washed in TBS/Tween 20, the membranes were incubated with HRP‐conjugated secondary antibodies for 1 hour at room temperature. The protein bands were visualized with ECL regents (Millipore, Boston, MA, USA).

Western blot analysis was performed as previously described with some modifications (47 (link)). Briefly, equal numbers of cells were collected when strains grew to the exponential phase in fermentation with acetic acid stress. Cells were treated with the following buffers: (i) distilled water, (ii) 2 M sodium hydroxide-8% mercaptoethanol, and (iii) TAP extraction buffer (4 mM HEPES-KOH [pH 7.5], 10% glycerol, 0.35 M NaCl, 0.1% Tween 20, 1× protease inhibitor cocktail, 1 mM PMSF, and 1× phosphatase inhibitor complex III [Sangon Biotech, China]). The sediment of cells was resuspended in 2× SDS loading buffer (0.1 M Tris-HCl [pH 6.8], 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 2% mercaptoethanol) and boiled for 10 min. Protein samples were separated by 12% SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membrane. The blots were probed with antibodies against specific proteins, followed by incubation with goat anti-mouse or goat anti-rabbit secondary antibodies. The antibodies used in this study are listed in Table S4. The specific proteins were visualized by super ECL detection reagent (Yeasen, China) in FluorChem FC3 (Proteinsimple, USA).