Hsp60

The following primary antibodies were used in this study: BrdU (Biorad, MCA2060 or Abcam, ab6326, 1:200 dilution); DNA (Progen, AC-30-10, 1:250 dilution); GAPDH (Sigma, G8795 or Abcam, ab8245, 1:10,000 and 1:2000 dilutions, respectively); GRP78 (Santa Cruz Biotech, Sc-13968, 1:1000 dilution); HSP60 (Abcam, ab46798, 1:1000 dilution); LC3B (Sigma, L7543) 1:5000; MTCO-2 (Abcam, ab110258 1:1000 dilution); NDUFB8 (Abcam, ab110242, 1:1000 dilution); AMPK alpha (Cell Signaling, 2532, 1:1000 dilution); phosphoAMPK alpha (Cell Signaling, 2531, 1:1000 dilution); TOM20 (Santa Cruz Biotech or Abcam, Ab186735, 1:4000 and 1:10,000 dilutions, respectively); VCL (Abcam, ab18058, 1:1000 dilution); and proliferating cell nuclear antigen (PCNA) (Mouse, sc-56, 1:8000 dilution).
Secondary Antibodies: Anti-Mouse IgG (H + L), HRP Conjugate (Promega, W4021, 1:4000 dilution); anti-Rabbit IgG (H + L), HRP Conjugate (Promega, W4011 1:4000 dilution); Alexa Fluor^®−488 goat- anti-mouse (Invitrogen, A-10684,1:1000 dilution); Alexa Fluor^®−568 goat- anti-mouse (Invitrogen, A-11004, 1:1000 dilution); Alexa Fluor^® 568 donkey anti-rabbit (Invitrogen, A-10042, 1:1000 dilution); Alexa Fluor^®-488 goat- anti-rat (Invitrogen, A-11006, 1:1000 dilution).

Fibroblasts were lysed in buffer containing (in mM): 25 Tris·HCl, pH 7.4, 150 NaCl, 1 sodium orthovanadate, 20 sodium fluoride, 10 sodium pyrophosphate, 2 EGTA, 2 EDTA, 1 phenylmethylsulfonyl fluoride, supplemented with 0.1% sodium dodecyl sulfate (SDS), 1.5% dodecyl-maltoside and a protease inhibitor cocktail [4 (link)]. The lysate was centrifuged for 30 min at 20,000 x g at 4°C to remove insoluble material, and the resulting supernatant was used for immunoblotting. Samples were electrophoresed on 8% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk (wt/vol) in TBST (in mM: 25 Tris-HCl pH 7.6, 150 NaCl and 0.05% Tween 20) and incubated with the following primary antibodies: LRPPRC (1/10 000), VDAC (1/1000, Abcam), HSP60 (1/10 000, Abcam), actin (1/10 000, Abcam). LRPPRC polyclonal antibodies were raised in rabbit against a 22 amino acid peptide having the sequence CEPPESFEFYAQQLRKLRENSS (antibody 295–313; Zymed Laboratories, San Francisco, CA). After washing, membranes were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase. Antigen-antibody complexes were detected by the enhanced chemiluminescence method. Quantitative analysis was performed with a GS-800 calibrated densitometer (Bio-Rad) using Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Proteins were separated using 18 well 4–20% Criterion TGX gels (Bio-Rad) and transferred to PVDF membranes using a Trans-Blot Turbo system (Bio-Rad). Western blotting was performed according to the protocol set forth by LI-COR for Near-Infrared Western Blot Detection. Incubations with primary antibodies were done overnight at 4 °C, while those with secondary antibodies were done for 1 h at RT. Blots were imaged using an Odyssey CLx system and quantification was done using instrument’s Image Studio software. The antibodies used were β-actin (Sigma A1978, 1:40,000–200,000), DJ-1 (Abcam ab76008, 1:4000–10,000), CRYAB (Abcam ab13496, 1:1000), HSP60 (Abcam ab46798, 1:10,000), GLUL (Millipore MAB302, 1:40,000), GAD2 (Cell Signaling 5843S, 1:4000), PHGDH (Atlas Antibodies HPA021241, 1:4000), NEFL (Cell Signaling 2835S, 1:4000), HK1 (Cell Signaling 2024S, 1:4000), pan AKT (Cell Signaling 2920S, 1:1000), pAKT S473 (Cell Signaling 4060S, 1:1000), pAKT T308 (Cell Signaling 13,038, 1:1000), GSK3β (Cell Signaling 9315, 1:1000), pGSK3β S9 (Cell Signaling 5558, 1:1000), HK2 (Cell Signaling 2867S, 1:2000), VDAC (Abcam ab14734, 1:2000), parkin (Cell Signaling 4211, 1:2000), PTEN (Cell Signaling 9556, 1:1000), and pPTEN S380 (Cell Signaling 9551, 1:1000).

Recombinant proteins were commercially purchased: Merlin, SETDB1, BRG1, RNA helicase A, CALR, and TPM3 were purchased from Signalway Antibody (Greenbelt, MD, USA); HSPA5, ACTG1, ENO1, NRAS, ERK2, p16, and GMPS were purchased from Lifespan Biosciences (Seattle, MA, USA); HSP60, HSP70, and TPI were purchased from Abcam (Boston, MA, USA); GNAS and P4HB were purchased from Aviva Systems Biology (San Diego, CA, USA); DNMT3A was purchased from Novus Biologicals (Centennial, CO, USA). The proteins’ purity was >90%. When coating onto the polystyrene 96-well plates, the proteins were diluted to a final concentration of 0.75 μg/mL in pH 7.2 phosphate-buffered saline (PBS) (Gibco, Waltham, MA, USA), each well was loaded with 100 µL of diluted protein. The 1:200 diluted serum samples were used as primary antibodies for 100 μL/well and stayed at 37 °C for 1.5 h. Next, the plates were rinsed with PBS 3 times, 1:1000 HRP-conjugated goat anti-human IgG was used as the secondary antibody for 100 µL/well and stayed at 37 °C for 1 h. After another 3 times of PBS washes, the chromogenic substrate ABTS (2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid)) (Alfa Aesar, Tewksbury, MA, USA) was applied and an average optical density (OD) of 405 nm was used for plate reading.

A list of plasmids is provided in Table S1. Plasmids were prepared by standard methods. Antibodies against the following proteins were purchased: UBQLN1/2 (Sigma, clone 5F5), UBQLN4 (Abcam ab106443), L9 and Tom20 (Santa Cruz Biotech., T-17 and FL-145), HA (Covance, clone 16B12), Rpt5 (Abcam ab22635), α7 (Enzo Life Sciences PW8110), FLAG (Sigma, clone M2), Hsc/Hsp70 (AssayDesigns, SPA-822), Hsp60 (Abcam ab46798), ClpP (Abcam ab124822), Actin-HRP (Sigma A3854), OxPhos cocktail (Abcam ab110411). FLAG-M2 Affinity resin was from Sigma, and GFP-trap from Chromotek. Antibodies to Bag6, TRC40, SGTA, TRAPα, GFP, and RFP have been described (Fons et al., 2003 (link), Mariappan et al., 2010 (link), Hessa et al., 2011 (link)). Anti-Myc was clone 9E10. Anti-HA used for IPs and blots (e.g., Figure 2A) was raised in rabbits against the KLH-HA peptide conjugate. Recombinant proteins were either purchased (His6-Ubiquitin, E1, and E2 from Boston Biochem) or expressed and purified from E. coli as described (Mateja et al., 2015 (link)).