Protein extraction reagent type 4

Total proteins were extracted using the Protein Extraction Reagent Type 4 (P4, Sigma‐Aldrich). Cells were sonicated with 10 pulses of 15 s at 45 W Labsonic 1510 Sonicator (Braun, Melsungen, Germany) and clarified by centrifugation for 10 min at 10 000 g. Protein concentration was determined by the Bio‐Rad Protein Assay, based on Bradford's method. Twenty micrograms of proteins were separated by SDS/PAGE (Novex Tris‐Glycine gels) according to the Laemmli protocol [22 (link)] and then transferred to nitrocellulose (0.22 μm; Bio‐Rad) or LF PVDF (0.45 μm; Bio‐Rad) by wet transfer and Towbin blotting buffer (50 mm Tris, 150 mm NaCl, 20% v/v methanol). Membranes were probed with the primary antibodies diluted in 5% w/v nonfat dry milk or 5% BSA in TBS‐T. The primary antibodies used in this study were anti‐HDAC2 (CST) and anti‐Lamin A/C (CST). The utilized secondary antibodies were Alexa Fluor 790 (Thermo Fisher Scientific). Immunoreactive bands were recorded using the enhanced chemiluminescence (Advansta, Menlo Park, CA, USA) or fluorescence acquisition by ChemiDoc Touch Imaging System (Bio‐Rad). The whole lane normalization (WLN) strategy was adopted in all western blot analyses using a trihalo compound for protein visualization [23 (link), 24 (link), 25 (link)]. Acquired images were analyzed by image lab software 5.2.1 (Bio‐Rad) [26 (link)].

Collected cells were lysed in Protein Extraction Reagent Type 4 (Sigma-Aldrich, Merck KGaA) with PhosStop phosphatase inhibitor and cOmplete protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA). The protein concentration of cell lysates was measured with the Bradford reagent (Bio-Rad Laboratories, Inc.). Fifty microgram of total protein was electrophoresed through 10% SDS-PAGE gels and was transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.) using an electrophoretic transfer chamber (Millipore, Temecula, CA, USA). The blots were incubated with primary antibodies overnight at 4°C followed by incubating with relevant HRP-conjugated secondary antibodies (Goat anti-mouse IgG antibody (Abcam ab6789) and goat anti-rabbit IgG antibody (Abcom ab97051)) for 1 hour at room temperature. Actin (sc-4778, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. The signal was detected using an ECL western blotting detection kit (Promega, Fitchburg, WI, USA).

All the materials were sampled from 3:00 pm and ground into fine powder with liquid nitrogen. 100 mg powder was used for total protein isolation with Protein Extraction Reagent Type 4 (Sigma-Aldrich, USA). Briefly, 1.2 ml of cold methanol (4 µl protease inhibitor cocktail/ml methanol) was added into 100 mg powder and then proceed followed the manual. Isolated total protein was loaded in NuPAGE 4–12% Bis–Tris Gel (Invitrogen, Carlsbad, CA, USA) and then stained with Coomassie blue after separation with MOPS SDS Running Buffer (Invitrogen, Carlsbad, CA, USA). Protein levels were normalized based on the staining signal. Separated proteins were transformed into PVDF membrane with transfer buffer (Invitrogen, Carlsbad, CA, USA) and the blot of primary and second antibodies was followed overnight and for 2 h respectively after washing with TBS and TBST solution. Membrane staining was performed with 1-Step NBT/BCIP (Thermo Scientific, USA). The primary antibodies specific to SUSIBA1 and 6-SFT were used^{8 (link)} with the same blot patterns as previous publication^{8 (link)}. The blot sizes of SUSIBA1 and 6-SFT are 30 kDa and 23 kDa respectively, and High-Range protein marker (Merk, Cat. GERPN756E) was used. Cropped gels and blots are displayed in Fig. 3b in order to improve the clarity and conciseness of presentation. Uncropped images are placed in Supplementary Fig. S2.

Cells were harvested before and 30 min, 4 h, and 24 h after changes in NaCl concentration (2500× g, 5 min, 4 °C). After cell harvest, samples were kept on ice, and all centrifugation steps were performed at 4 °C. Cell lysis and protein extraction were performed using the Protein Extraction Reagent Type 4 (Sigma-Aldrich, St. Louis, MO, USA) (1:3, v/v). For purification and precipitation, four times the volume of Methanol (with 10 mM Diethiothreitol (DTT)) was added to the samples. After vortexing, the initial volume of chloroform was added, and the mixture was vortexed again. Three times the volume of bidest. water was added to the mix. The mixture was centrifuged after an additional vortexing step (15,000× g, 1 min). The top aqueous layer was removed, and four times the volume of methanol (with 10 mM DTT) was added and vortexed. After centrifugation, the supernatant was removed, and the pallet was washed with acetone. The pellet was air-dried and dissolved in 8 M urea solution supplemented with 10 mM DTT. Three biological and three technical replicates were prepared for every condition.