Spin column

Protein samples were thawed and mixed using a spin column (Bio-Rad), and then separated on a 5% SDS-PAGE gel for Western blot analysis. After gel electrophoresis, proteins were transferred to nitrocellulose (NC) membranes (Millipore Corporation, Billerica, MA, USA). The membranes containing protein were blocked with 5% fat-free milk in TBST at room temperature (18~24 °C) for 2 h, and then hybridized using anti-Hsp70-and-Hsp90 antibody (1:1000, Abcam, Hong Kong) or rabbit polyclonal antibody (1:2000, Abcam, Hong Kong) at 4 °C overnight. The membrane was then washed four times with TBST and labeled with HRP-conjugated secondary antibody (1:4000,) for 2 h at room temperature (18~24 °C). After washing five times with 1× TBST, Hsp70 and Hsp90 were detected on the membrane with an ECL detection kit (Beyotime, Shanghai, China). The protein expression intensity was determined by optical density analysis. The intensities of β-actin bands were used for the single standard [20 (link)].

The cells were cultured in three 15 cm culture dishes to ~ 80% confluency for each cell line in triplicates. Then they were harvested by a cell scraper, centrifuged (1000 g, 4 °C, 5 min), washed with PBS, and the cell pellets were frozen at − 80 °C. The frozen cell pellet was resuspended with HNN-lysis buffer (0.5% NP40, 200 mM Na₃VO₄, 1 mM PMSF, 1.2 μM avidin, Complete protease inhibitors without EDTA (Roche, Switzerland)) and subsequently pipetted to dissolve and avoid foaming. The suspension was incubated on ice for 10 min, then transferred to a 2 ml microtube and centrifuged at 14,000 g for 20 min at 4 °C. 250 μl of lysis buffer was added to Bio-Rad Spin Column (Bio-Rad, cat. No 732-6008) to avoid the formation of air bubbles. 100 μl of High Capacity Streptavidin Agarose Resin (Thermo Fisher Scientific, cat. No 20359) were mixed in 750 μl of HNN-lysis buffer, and 200 μl of the prepared beads were added to samples which were then incubated for 15 min at 4 °C on a rotary wheel. The beads were then washed twice with 1 ml of HNN-lysis buffer using Bio-Rad Mini Columns. After washing with lysis buffer, samples were washed three times with 1 ml of HNN buffer (50 mM HEPES, 150 mM NaCl, 50 mM NaF), which contained no detergent and inhibitors. Finally, samples were eluted with 200 μl of 2.5 mM Biotin in HNN buffer three times.

RNA was isolated by following the protocol previously described [4 (link)]. The isolated RNA was purified using Bio-Rad spin column (Cat. No. 732–6250, Bio-Rad, Hercules, CA) and treated with DNase I (Cat. No. R1013, Zymo Research, Irvine, CA) to remove any genomic DNA contamination. The integrity and quality of RNA samples were analyzed on Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) using RNA 6000 Pico kit (Cat. No. 5067–1513, Agilent Technologies, Santa Clara, CA). Bacterial rRNAs were removed using Life technologies MICROBExpress kit (Cat. No. AM1905, Grand Island, NY) and the extent of the removal of rRNA from the samples was analyzed on Agilent Bioanalyzer 2100.

For affinity purification, tetracycline-inducible Flp-In T-REx 293 cell lines expressing KCNQ1 wild type and other mutations were generated as described^{56 (link)}. In brief, the cells were co-transfected with the tagged-KCNQ1 expression constructs and pOG44 vector (for Flp-recombinase expression, Invitrogen) using FuGENE 6 transfection reagent (Promega). After 2 days, transfected cells were selected in Hygromycin B (100 mg/ml; Invitrogen) containing medium for 3 weeks. The positive clones containing stable isogenic tagged-KCNQ1 (wt and mutants) were expanded and for each construct ∼5 × 10⁷ cells (5 × 15 cm dishes) in three biological replicates were induced with 1 μg/ml doxycycline (MP Biomedicals) for 24 h. After induction, cells were washed with 0.1 mM MgCl₂, 0.1 mM CaCl₂ in PBS and harvested in 1 mM EDTA-PBS. Cells were pelleted by centrifugation 400 × g, 5 min, 4 °C. Cell pellets (~5 × 10⁷ cells) were lysed in 3 ml of HENN-lysis buffer (50 mM HEPES pH 8.0, 5 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.5% NP40, 1 mM DTT, 1.5 mM Na₃VO₄, 1 mM PMSF, and 1× protease inhibitors cocktail; Sigma) on ice. Cleared cell lysates were loaded on spin columns (Bio-Rad, USA) containing 200 ml of Strep-Tactin beads (IBA GmbH), washed thrice with 1 ml HENN-lysis buffer and 4× with 1 ml HENN buffer. Bound proteins were eluted with 900 μl of elution buffer (0.5 mM Biotin; Pierce).