Hplc station

The following reagents were used: Matrigel (BD Bioscience), DAPI/Antifade solution (CHEMICON International, Inc), Cell Tracker Green CMFDA (Invitrogen, Tokyo, Japan), Vybrant FAM caspase-3 and -7 assay kit (Molecular Probes), MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Sigma-Aldrich), DAPI/Antifade solution (Millipore, Temecula, CA), chloroquine (Tocris Bioscience), complete protease inhibitor cocktail (Roshe Diagnostics,Germany), PMSF (Sigma-Aldrich). Molecular weight markers were from Thermo Scientific: 14.4–116.0 kDa (#26610) and 10–250 kDa (#26619).
Antibodies against human LC3A/B, MDM2, Bax and tubulin were from Abcam, p53 and Bcl-2 were from Sigma-Aldrich, and anti-RL2 was from BioSan-R (Novosibirsk, Russia). The secondary antibodies used in this study were HRP-conjugated goat anti-mouse and goat anti-rabbit (BioSan-R, Novosibirsk, Russia) or FITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich).
The recombinant analogue of lactaptin RL2 was obtained from E.coli and purified as described previously [3] (link). The 98% purity of the isolated protein was confirmed by RP-HPLC chromatography on C5 reverse phase column (Discovery BIO Wide Pore C5, Sigma) in water (0.5% TFA)-acetonitil solvent system using HPLC Station (Bio-RAD Laboratories) as well as by RP-HPLC on C18 (ProntosSIL) using Milichrom A-02 station (EcoNova, Russia).

Heparin sodium salt (H4784-1G) and CaCl₂ were from Sigma-Aldrich. DNA Gel Loading Dye (R0611) and Lipofectamine 3000 were from ThermoFisher, molecular weight markers Sky High (10 and 500 bp) were from BioLabMix, Russia. MTT (3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide was from Sigma-Aldrich. Plasmid pEGFP-C1 (GenBank Accession U55763; cat 6084-1) was purchased from BD Biosciences Clontec, monoclonal anti-RL2 mouse IgG (clone F14; Biosan, Russia).
The recombinant analogue of lactaptin RL2 was obtained from E. coli and purified as described previously (Semenov et al., 2010 (link)). The 98% purity of the isolated protein was confirmed by RP-HPLC chromatography on C5 reverse phase column (Discovery BIO Wide Pore C5; Sigma) in water (0.5% TFA)-acetonitil solvent system using HPLC Station (Bio-RAD Laboratories) as well as by RP-HPLC on C18 (ProntosSIL) using Milichrom A-02 station (EcoNova, Russia) (Supplementary Figure 3A) and by SDS-PAAG electrophoresis under reduction conditions (Supplementary Figure 3B).