Eps15

All cell culture reagents were purchased from Gibco-Invitrogen (Carlsbard, CA, USA). Cell culture dishes and other plasticware were obtained from Nalgene Nunc (Rochester, NY, USA). Salts and buffers were purchased Sigma (St. Louis, MO, USA). Leptomycin B (LMB), α-tocopherol, LPS O111:B4 and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) were obtained from Sigma-Aldrich. Primary antibodies used in protein detection of CALM, α-adaptin, β-adaptin, eps 15, c-Myc, c-Jun, c-Abl and SR-B1 as well as secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Abcam (Cambridge, UK). Fluorescent conjugates of LPS BODIPY FL, and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate for ROS detection were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Antibodies used in this study for immunofluorescence staining include: clathrin heavy chain mouse monoclonal (clone X22, 1:250 dilution), AP2 α‐adaptin mouse monoclonal (clone AP6, AbCam ab2730, 1:200 dilution), clathrin heavy chain rabbit polyclonal (AbCam, ab21679, 1:1500 dilution), Eps15R rabbit polyclonal (AbCam, ab53006, 1:200 dilution), FCHo2 rabbit polyclonal (Ra103; aa 525‐890, 1:200 dilution31), Eps15 rabbit polyclonal (Ra15, aa 530‐791; 1:500 dilution62). Goat anti‐mouse and anti‐rabbit Alexa‐488, ‐546 and ‐647 conjugated secondary antibodies were used at 1:500 dilution (Life Technologies). For immunoblotting the following antibodies were used: clathrin heavy chain (BD Transduction Laboratories, #610500, 1:10 000 dilution), AP2‐β‐adaptin (clone 100/1, Sigma, A4450, 1:5000 dilution), EGFP (AbCam, ab290, 1:20 000 dilution), Flag (clone M2, Sigma, F3165, 1:2000 dilution), His (GE Healthcare, #27‐4710‐01, 1:1000 dilution), β‐actin (AbCam, ab6276, 1:10 000 dilution), Eps15 (Santa Cruz, C‐20, 1:2000 dilution).

Proteins were resolved on either 10% Tris-glycine gels or 4–20% Novex Tris-glycine gels (Life Technologies) and transferred onto nitrocellulose or PVDF membranes. Immunoblots were probed for HGS, STAM1, TSG101, EPS15, EGFR (Santa Cruz, Dallas, TX), TrkA and TrkB (Millipore, Billerica, MA), CHMP2B (Abcam, Cambridge, MA), ERBB2, p62, LC3 (4108) and cleaved caspase 3 (Cell Signaling, Danvers, MA), mylein basic protein (Biolegend, Dedham, MA), GFAP (Dako, Carpinteria, CA) and ubiquitin (UAB Hybridoma Facility, Birmingham, AL). β-tubulin and β-actin (Developmental Hybridoma Bank, Iowa City, IA) were used for loading controls. Primary antibodies were diluted in 1X phosphate buffered saline containing 0.1% NP-40 and either 2% BSA or 1% non-fat dry milk, and proteins were detected by using an anti-mouse or anti-rabbit HRP-conjugated secondary antibody (Southern Biotechnology Associates, Birmingham, AL) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL).