Semi dry apparatus

Protein extraction and western blotting were performed as previously described[14 (link)]. Cell lysates were extracted with the T-PER tissue protein extraction reagent (Pierce, Rockford, IL) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science). Equal amounts of total proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane using a Bio-Rad SemiDry apparatus. After blocking for nonspecific binding, the membranes were incubated with anti-CLU (1:200 dilution; Santa Cruz Biotechnology), MMP13 (1:200 dilution; Santa Cruz Biotechnology), p-Akt (1:1000 dilution; Cell Signaling Technology), Akt (1:1000 dilution; Cell Signaling Technology), EIF3I (1:800 dilution; Abcam, Hong Kong, China) or GAPDH (1:5000 dilution; Kang-Chen, Shanghai, China) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. After washing three times in TBST, protein bands were visualized using chemiluminescence detection.

Microsomal fractions were isolated as previously described (Jasiński et al., 2001 (link)) from 150 mg of Medicago hairy roots or 300 mg of BY2 cells. The proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore) by electroblotting (semi-dry; apparatus; Bio-Rad). The membrane was incubated with a primary polyclonal antibody specific for MtABCG10 (Banasiak et al., 2013 (link)) or with a primary antibody against H+-ATPase (W1G) (Morsomme et al., 1998 (link)). The secondary antibody was an alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma).

The dorsal skin was collected and was treated with 2.5 U/mL Dispase® II (neutral protease, grade II) in PBS solution overnight at 4°C. After the incubation time, the epidermis and dermis layers were gently separated using a scalpel. The dermis layer was stored at −80°C before use. The proteins were extracted from the dermal layer using RIPA buffer that is consist of protease cocktail inhibitor (1 μg/1 μL). The cytosolic and nuclear extracts were prepared by the method previously described [27 (link)]. The amounts of protein concentration were measured by using the NanoDrop spectrophotometer (Thermo Scientific, Austria). Briefly, 50 μg of protein samples from each group was fractionated on 8-10% SDS-PAGE gel, and it was transferred to a nitrocellulose membrane (Bio-Rad, Germany) using a semidry apparatus (Bio-Rad). The membrane was blocked with 5% nonfat milk (blocking solution) for 1 h at 4°C; then, respective primary antibody (1 : 1000 in 5% BSA solution) was added and incubated overnight at 4°C. The membranes were washed with TBST buffer for 15 min intervals and then incubated with horseradish peroxidase-conjugated secondary antibody (1 : 5000 in nonfat milk) and kept for incubation for 1 h at 37°C. After the TBST buffer washing, the protein bands were spotted using a chemiluminescence detection method and the images were quantified by using Image Studio software (LI-COR).

Total RNA (15 μg) was isolated using Trizol (Life Technologies) and resolved on a denaturing 8 M UREA/15% polyacrylamide (19:1, acrylamide:bisacrylamide) 1 × TBE gel. The gel samples were transferred to a Hybond N+ membrane (GE Healthcare) using a semi-dry apparatus (Bio-Rad) at room temperature at 300 mA for 1.5 h in 1 × TBE transfer buffer. Following transfer, the RNA was crosslinked to the membrane using a Stratalinker UV Crosslinker (Stratagene). The membrane was pre-hybridized in buffer containing 0.2 M Na₂PO₄ buffer (pH 7)/ 7% SDS for 30 min at 37 °C, at which point 40 × 10⁶ c.p.m. of radioactive probe was added and incubated overnight at 37 °C. Probes were labelled using T4 PNK (NEB) and γ-³²P-ATP (Perkin Elmer) and unincorporated γ-³²P-ATP removed by G-25 Sephadex spin columns. The following probes were used: U6 (loading control)—5′-GCAGGGGCCATGCTAATCTTCTCTGTATCG-3′ and gRNA (targeting the scaffold portion of the gRNA) 5′-TTCAAGTTGATAACGGACTAGCCT-3′. After incubation, the membrane was washed twice with 2 × SSPE/0.1% SDS for 30 min. at 42 °C and twice with 0.5 × SSPE/ 0.1% SDS for 30 min at 42 °C. The membrane was exposed to a Phosphorimager screen for a few hours and processed using a Typhoon scanner (GE Healthcare) or exposed overnight at −80 °C to X-ray film. Quantitation was performed using ImageJ (NIH). Full uncropped images are provided in Supplementary Fig. 6.

Cell samples were harvested by scraping, washed twice with cold PBS, lysed in lysis buffer (150 mM Tris, pH 7.5, with 150 mM NaCl, 1% Triton X-100, and complete protease inhibitor cocktail tablets [Roche]) at 4 °C for 30 min, and centrifuged at 10,000 g for 30 min. The supernatants were mixed with 1X loading buffer (0.08 M Tris, pH 6.8, with 2.0% SDS, 10% glycerol, 0.1 M DTT, and 0.2% bromophenol blue) and boiled for 5 min. The cell lysates were separated via SDS-PAGE and transferred to nitrocellulose membranes using a semidry apparatus (Bio-Rad). The membranes were probed with various primary antibodies against the proteins of interest; secondary antibodies were alkaline phosphatase-conjugated anti-goat IgG and anti-mouse IgG (Jackson ImmunoResearch Laboratories). Staining was conducted with 5-bromo-4-chloro-3-indolyl phosphate and NBT solutions prepared from chemicals obtained from Sigma-Aldrich (Milwaukee, USA). The uncropped blots are provided as a Source Data file.