Mouse anti ubiquitin fk2

Protocols have been extensively described recently (Lucocq et al., 2001 (link), Karanasios and Ktistakis, 2015 (link)). In brief, cells on coverslips were fixed in 3.7% Formaldehyde, permeabilized in 0.1% NP40 and stained in a blocking solution containing fish gelation and 0.05% NP40. Antibodies used and their final dilution are as follows: rabbit anti-OPTN (Cayman Chemicals), 1:100; mouse anti-WIPI2 (Bio-Rad), 1:200; rabbit anti-phospho(S172)TBK1 (Cell Signalling), 1:50; mouse anti-Ubiquitin FK2 (Enzo), 1:100; rabbit anti-FIP200 (ProteinTech), 1:100; rabbit anti-ATG9 (Cell Signalling), 1:100; mouse anti-TOMM20 (Abcam), 1:100; rabbit anti-ATG13 (Sigma), 1:100; rabbit anti-LC3 (Sigma), 1:150; rabbit anti-NDP52 (GeneTex), 1:100; rabbit anti-Tax1BP1 (ProteinTech), 1:100; rabbit anti-VAPA (Sigma), 1:200; rabbit anti-VAPB (Sigma), 1:200; mouse anti-EEA1 (BD Biosciences), 1:70; mouse anti-LAMP2 (Developmental Studies Hybridoma Data Bank), 1:200; mouse anti-TUBULIN (Sigma), 1:300; mouse anti-cytochrome C (Abcam), 1:200; rabbit anti-phospho(S351)p62 (kind gift from Dr Masaaki Komatsu), 1:100; rabbit anti-TRAF2 (Abcam) 1:100.

The following antibodies were used: mouse anti-Flag M2 (Sigma, A8592), mouse anti-ubiquitin FK2 (Enzo, PW8810), rabbit anti-ubiquitin (Dako, Ely, UK; Z0458), mouse anti-p97 (Fitzgerald, North Acton, USA; 10R-P104A), rabbit anti-VAPA (Epitomics, Burlingame, USA; S1706), mouse anti-FAF1 (Abnova, Taipei City, Taiwan; H00011124-A01), rabbit anti-FAF1 (courtesy of Millipore, Billerica, USA), rabbit anti-GAPDH (Cell Signaling, 2118), rabbit anti-RAB3GAP1 (Proteintech, Manchester, UK; 21663-1-AP), rabbit anti-RAB3GAP2 (Abgent, Maidenhead, UK; AP9635B), rabbit anti-WDR44 (Bethyl, Montgomery, USA; A301-441A), mouse anti-ASNA1 (Abnova, H00000439-M03), rabbit anti-Syntaxin 1A (GeneTex, Irvine, USA; GTX113559), rabbit anti-BAG6 (Cell Signaling, 8523S), mouse anti-RPN2 (Abnova, H00006185-B01), rabbit anti-CD147 (Abcam, Cambridge, UK; ab108317), Alexa Fluor® 488 goat anti-rabbit (Invitrogen, A11008) and Alexa Fluor® 594 chicken anti-mouse (Invitrogen, A21201). Anti-VAPA (Epitomics, S1706) antibodies recognize both VAPA (upper band) and VAPB (lower band) in human cell extracts. The following antibodies were raised and affinity purified using the appropriate antigen by the DSTT: sheep anti-FAF1 (S370D; antigen human FAF1, 1-650), rabbit anti-VAPB (R2986 and R2987; antigen human VAPB, 1-210).

Cav1-GFP, Cav1-mCherry, and Cav1-myc were as described previously15 (link)16 (link). Details of different Cav1 constructs used are described in Supplementary Table S1. Rabbit anti-Cav1 polyclonal antibody (catalog number 610059) and mouse monoclonal (mAb) anti-Cav1 clone 2234 (mAb 2234, catalog number 610494) were purchased from BD Biosciences. Mouse anti-Myc (9B11) was procured from Cell Signaling. Rabbit Anti-Giantin (# ab24586) was purchased from AbCam, whereas mouse anti-Golgin97 (Clone CDF4) was obtained from Thermo Scientific. Mouse anti-Vimentin (# V6389) was purchased from Sigma. Chicken anti-vimentin (Clone Poly 29191) was purchased from Biolegend. Mouse anti-Ubiquitin (Fk2) (# BML-PW8810-0500) and anti-20S proteasome antibodies (# BML-PW8115-0025) were procured from Enzo Life Sciences. Mouse anti-VCP/p97 (# NB120-11433) antibody was purchased from Novus Biological. Anti-GFP antibody (Clone JL8) was purchased from Geneclone. Anti-Beta tubulin antibody E7 was procured from DSHB, UIOWA. MG132, Nocodazole, DMSO and chloroquine were purchased from Sigma. Cycloheximide was procured from MP Biomedicals. VCP/p97 inhibitor DBeQ was procured from Tocris. Lipofectamine 2000 and Prolong Gold anti-fade mounting reagent were obtained from Life Technologies.