Myc tag

Immunoblotting was performed using standard methods and visualized by enhanced chemiluminescence as previously described (81 (link)). Antibodies were used at the following concentrations: CLVS1 (C82727 Lifespan Biosciences and PA5-32088 Invitrogen) 1:500, β-actin (6609 Proteintech) 1:3000, Myc-tag (PA1-981 Invitrogen) 1:800, DYKDDDDK (D6W5B Cell Signaling Technology) 1:800. Uncropped Western blots are shown in Supplemental Figure 10.

The following antibodies were used: EGFR (sc-03-G), Akt1 (sc-1618), C14ORF37 (UT2) (sc-139226), Rictor (sc-271081), Raptor (sc-81537) pAMPK (S485/491), AMPK (sc-25793), Alpha-Tubulin (sc-5546), were purchased from Santa Cruz Biotechnology. pEGFR (Y1173) from Invitrogen, Myc-tag (Cat#2278S), mTOR (Cat#2972), phospho p70S6K1 (T389) (Cat#9204), pPKC (T514) (Cat#9379), pAkt (S243) (Cat#4060), phospho MAPK (Cat#9101S), MAPK (Cat#4695), ULK1 (Cat#8054), pULK1-S757 (Cat#6888), and pULK1-(Cat#5869) were purchased from Cell Signaling. Actin (Cat#A2228) was from Sigma. PKC (Cat# ab71558) and phospho-mTOR(Cat#ab109268) were from Abcam. C225 (Cat #MABF120), was from EMD Millipore for EGFR immunoprecipitation studies. LC3 (Cat#NB100-2220) was from Novus biologicals. EGFR TKI AEE788 (Cat# S1486), Akt inhibitor, MK2206 (Cat#S1078), were obtained from Selleckchem. Plasmid-based transfections and siRNA-based transfections were performed using Lipofectamine 3000 (Invitrogen) and Lipofectamine RNAiMax (Invitrogen), respectively. Protein A/G beads (Santa Cruz Biotechnology) were used for immunoprecipitation.

DDIAS (1–998 amino acids) and HSP70 (1-641 amino acids) cDNA clones were obtained from the Korea Human Gene Bank, Medical Genomics Research center, KRIBB, Korea. DDIAS Full-length F (aa 1–998) and domain N (aa 1–400), M (aa 401–600) and C (aa 601–998) were amplified by PCR and cloned into the p3xFLAG-CMV vector (Sigma-Aldrich) to create the p3xFLAG-CMV-DDIAS. HSP70-wt (1-641 amino acids) clone was inserted into pcDNA3 vector with Myc tag (Invitrogen). The coding region of CHIP was obtained by PCR-amplification of HEK293T cDNA. HA-CHIP-wt (1-303 amino acids), HA-CHIP-ΔTPR (128–303 amino acids) and HA-CHIP-ΔUbox (1–195 amino acids) were amplified by PCR and inserted into the pcDNA3. Transfections were performed using TurboFect (Thermo Scientific, Rockford, lL, USA) according to the manufacturer's instructions.

The wild type (wt) MDA-9/Syntenin cDNA (Gene ID: 6386) was subcloned into SalI and NotI sites of the phosphorylated cytomegalovirus pCMV/myc/cyto plasmid, which has an N-terminal myc tag (Invitrogen) as described [4 (link)]. For knock down (KD) experiments, MDA-9/Syntenin-specific shRNA was constructed and cloned into the BamI-HindIII sites of pRNA3.1 vector (Genescript Corporation, Piscataway, NJ) as described earlier. Various MDA-9/Syntenin deletion constructs were also described previously [4 (link)].

Using full-length mouse MG53 DNA (GenBank accession number: NM_001079932) as a template, GST-fused full-length MG53 or MG53 domains were constructed and were expressed in E. coli (DH5α), as previously described30 (link)44 (link). GFP-tagged full-length MG53 or PRY-SPRY was constructed using an eGFP vector, as previously described16 (link). HA-MG53 was constructed, as previously described23 (link). Orai1-myc was constructed by inserting Orai1 DNA (GenBank accession number: NM_175423.3) into the 5′ end of pcDNA3.1 A containing a myc-tag (Invitrogen, Waltham, MA, USA)23 (link).

Cultured HUVECs were collected and lysed with lysis buffer containing 98% RIPA lysis buffer, 1% protease inhibitor, and 1% phosphatase inhibitor. The cells were incubated with the lysis buffer on ice for 30 min. The total protein concentration in the lysate was measured using the BCA protein assay kit from Beyotime Biotechnology, Shanghai, China. Equal amounts of protein from each sample were loaded onto SDS-PAGE gels and separated by electrophoresis. The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes and blocked with PBST containing 5% skim milk blocking buffer at room temperature for 2 h.
Primary antibodies, including O-GlcNAc (Cell Signaling Technology, #9875), GSDMD (Santa Cruz Biotechnology, sc-393581), GSDMD (Abcam, ab209845), β-actin (SAB, #21338), GAPDH (Proteintech), OGT (Santa Cruz Biotechnology, sc-74546), Myc Tag (Invitrogen, # R951-25), OGA (Proteintech, 14711-1-AP), were incubated with the membranes followed by incubation with HRP-conjugated secondary antibodies (anti-rabbit IgG from Bioword, and anti-mouse IgG from Bioword). Protein bands were detected using an Enhanced Chemiluminescent (ECL) reagent from Thermo Scientific, and the images were acquired with a ChemiDoc MP System from Bio-Rad. Densitometric analysis was performed using ImageJ Software to quantify the protein bands.