Anti ha

Cell lysates were prepared using high KCl lysis buffer containing 10 mMTris-HCl, pH 8.0, 140 mMNaCl, 300 mMKCl, 1 mM EDTA, 0.5% TritonX-100 and 0.5% sodium deoxycholate with complete protease inhibitor cocktail (Roche) and 20 mM N-ethylmaleimide (NEMI) to block free thiol groups. Equal amounts of proteins (30 μg) were subjected to SDS-PAGE with loading buffer with or without β-mercaptoethanol and transferred to polyvinylidene fluoride (PVDF) membranes (Roche). The membranes were treated with 1% blocking solution in TBS for 1 h, immunoblots were probed with the indicated antibodies: anti-GFP (1:5,000; AbMart, Shanghai, China), anti-Flag (1:5,000; AbMart, Shanghai, China), anti-HA (1:5,000; AbMart, Shanghai, China), anti-Prdx-SO₃ (1:2,000; Abcam) at 4 °C overnight. Then the membranes were washed and incubated with POD-labeled secondary antibodies (1:125,000; Roche). The immunolabeled proteins were detected by BM Chemiluminescence Western Blotting kit (Roche).

Proteins from the sample lysate were fractionated by SDS–PAGE. The separated proteins were transferred from the gel to an Immobilon-PSQ polyvinylidene difluoride membrane (pretreated with methanol for 15 s; Millipore) using a transfer buffer (20 mM Tris, 150 mM glycine). The membrane was then blocked using PBS(pH 7.4) containing 3% non-fat dry milk for 30 min at room temperature with 50 r.p.m. shaking, followed by one wash with PBST (PBS with 0.1% Tween 20). Anti-Flag (1:2,000; #F3165; Sigma-Aldrich) and anti-BiP (1:1,000; #sc-33757; Santa Cruz Biotechnology), anti-GFP (1:2,000; #M20004; Abmart), anti-RFP (1:1,000; #5f8; Chromotek), anti-HA (1:2,000; #M20003; Abmart), anti-myc (1:2,000; #M20002; Abmart) and anti-actin (1:2,000; #M20009; Abmart) antibodies were added to PBSTM (PBS with 0.1% Tween 20 and 3% non-fat dry milk) and incubated at room temperature for 90 min, followed by three washes (5 min each) with PBST. The membrane was then incubated with a goat anti-mouse IRDye 800CW antibody (Odyssey, no. 926-32210; Li-Cor) at a ratio of 1:10,000 in PBSTM at room temperature for 30 min with 50 r.p.m. shaking. The membrane was washed three times (5 min each) with PBST, once for 5 min with PBS, and then visualized by excitation at 780 and 800 nm. Full-size images are presented in Supplementary Fig. 14.

The protein stability assay was performed with a transient expression system in N. benthamiana. The 35S::PSY1-HA and 35S::Flag-PPSR1 constructs were generated as described above. The resulting constructs were individually introduced into A. tumefaciens strain GV3101^{58 (link)}. After cultivation, the Agrobacteria harboring the indicated constructs were infiltrated into N. benthamiana leaves^{64 (link)}. Forty-two hours after infiltration, the leaves were treated with 50 µM MG132 or DMSO (negative control) for 6 h. The total proteins were then extracted from the N. benthamiana leaves as described above and subjected to immunoblot using anti-HA (Abmart), and anti-Flag (MBL Life Science) antibodies, respectively. Equal loading was confirmed with an anti-actin antibody (Abmart).
For degradation rate analysis, the agroinfiltrated N. benthamiana leaves were treated with 250 μM cycloheximide (CHX) after 36 h of incubation. The mutated form of PSY1 (PSY1^K380R, PSY1^K406R, and PSY1^K380/406R) was generated by site-directed mutagenesis using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies) following the manufacturer’s instructions. The band intensity was quantified using ImageJ software (https://imagej.nih.gov/ij/index.html). Values represent the average of three independent replicates. The primers used for vector construction are listed in Supplementary Data 3.

A plasmid containing wild-type human SMYD4(BC035077) was obtained from Abmgood (Abmgood, USA). Then, Flag-tagged wild-type SMYD4 and HA-tagged wild-type HDAC1 were cloned into the expression plasmid. The mutation (G345D) identified in patients was obtained using the KOD-Plus Mutagenesis Kit (Toyobo, Japan). All plasmids were confirmed via Sanger sequencing. The anti-SMYD4 (Proteintech, USA), anti-HDAC1 (Proteintech, USA), anti-Flag (Abmart, China), and anti-HA (Abmart, China) antibodies were used. Wild-type SMYD4 was overexpressed in HL-1 cells. After 48-h transfections, cell lysates were obtained in RIPA containing 1 mM PMSF and complete protease inhibitors (Roche, USA). Immunoprecipitation was performed using an anti-Flag affinity gel (Biotool, USA). SDS-PAGE was performed to resolve the eluates. After sliver staining, the proteins underwent mass spectrometry analysis. Protein-protein interactions were verified in HL-1 cells after transient overexpression of SMYD4. Co-immunoprecipitation was performed to confirm the interaction between SMYD4 and HDAC1 in HEK293T cells using anti-tag antibodies.

Immunostaining analyses were performed as previously described (Jiang et al., 2017 (link)). In brief, transfected HEK293T cells were washed once with phosphate-buffered saline (PBS; Thermo Scientific), fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, and washed three times with PBS. Then, cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature for total protein analysis or were left unpermeabilized for surface protein analysis. After blocking with PBS containing 5% milk and 3% goat serum for 30 min at room temperature, cells were incubated with a primary antibody (anti-HA, 1:1000, Abmart; anti-Flag, 1:1000, Abmart) for 2 h at room temperature, washed three times with PBS, and incubated with the secondary antibody (donkey anti-mouse Alexa Fluor 546-conjugated secondary antibody, Life Technologies) for 30 min at room temperature. Fluoromount-G (Southern Biotech) was used to mount the cells on microscope slides. Images were acquired with a laser scanning confocal microscope (Olympus, FV3000) using a 60× objective lens (Olympus) and were further analyzed using the National Institutes of Health ImageJ program and Prism 5 software (GraphPad Prism).

Mouse primary hepatocytes were transfected with the indicated plasmids and lysed in 10% SDS lysis buffer, then denatured by heating at 95 °C for 10 min and IP lysis buffer (20 mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; and 1% NP-40) were added to the lysates. After sonication and centrifugation (12,000g for 10 min), the supernatants were incubated with anti-HA (Abmart, Cat#M2003S,) or anti-PEPCK1 antibody (Proteintech, Cat#16754-1-AP) as well as protein A/G agarose beads at 4 °C for 4 h. The beads were then washed with IP lysis buffer three times. Furthermore, the protein was boiled with SDS loading buffer after centrifugation for 10 min. Finally, western blotting was performed.

Semi-in vivo pull-down assay was conducted as described by Hu et al.^{68 (link)} with some modifications. The coding region of PSY1 was amplified from tomato cDNA and cloned into the pCambia1300-MCS-HA vector to generate 35S::PSY1-HA construct. The resulting construct was transformed into A. tumefaciens strain GV3101^{58 (link)}, which was subsequently infiltrated into N. benthamiana leaves^{64 (link)}. After infiltration for 48 h, the N. benthamiana leaves were collected. Total proteins were extracted from the leaves with 1 ml of extraction buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5% glycerol, 1 mM PMSF, 1× protease inhibitor cocktail, and 50 μM ΜG132. After centrifugation at 12,000 × g for 20 min at 4 °C, the supernatant containing HA-tagged PSY1 (PSY1-HA) was collected and mixed with 500 ng of recombinant MBP-PPSR1 or MBP (negative control). Then the mixture was incubated with anti-HA agarose (Cell Signaling Technology) at 4 °C for 2 h. The agarose beads were collected and washed three times with extraction buffer. The proteins eluted from the beads were then subjected to immunoblot with anti-MBP (Beijing Protein Innovation) and anti-HA (Abmart) antibodies, respectively, as below.