Bml pw8810

Immunostaining of Drosophila larvae was carried out as described previously [19 (link)]. Briefly, L3, or late L2 for analysis of autophagic flux, larvae were dissected in chilled in Ca⁺²-free HL3 solution [33 (link)], fixed in 4% formaldehyde in PBS for 30 min, permeabilised in 0.1% Triton X-100 in PBS and incubated with primary antibodies against HA (as above), Dlg (4F3, Hybridoma; Iowa City, IA, USA), GFP (A-6455, Thermo Fisher Scientific; USA), HRP (P7899, Sigma) and FK2 (BML-PW8810, Enzo Life Sciences; Farmingdale, NY, USA). Following incubation with the appropriate secondary antibodies, fixed preparations were mounted in Vectashield containing the nuclear stain DAPI (Vector Laboratories; Oxfordshire, UK) and were viewed using an Olympus IX81 confocal head mounted on an Olympus Fluoview FV-1000 microscope. All images were acquired using a 60×/1.35 NA objective and FV10-ASW cer.04.01 software. All axonal analyses were conducted by imaging motor axon bundles passing through segment A7. Similarly, analyses of neuromuscular junctions (NMJs) were conducted by imaging muscles 6 and 7 at segment A7. Z-stacks of larval fat bodies were imaged at approximately 2-μm intervals and were subsequently converted into maximum intensity projections for image analysis.

nda3-Km311 cells were incubated at the restrictive temperature (18 °C), and proteasome inhibitors Bortezomib (Selleckchem, #S1013) and MG132 (MedChem Express, #HY-13259) were added directly to the culture to a final concentration of 250 μM and 50 μM respectively. PR-619, a reversible inhibitor of deubiquitinylating enzymes (Selleckchem, #S7130), was also added to a final concentration of 5 μM. Additionally, lysis buffer used for extract preparation was supplemented with 50 μM MG132, 50 μM PR-619, and 20mN of the inhibitor of cysteine peptidases NEM (Selleckchem, #S3692).
Alternatively, mts3-1 cells expressing Slp1-HA were grown in YES to mid-log phase at permissive (25 °C) temperature and then shifted to restrictive (36 °C) temperature for 3 h. 0.6 M KCl was added to the cultures 30 min before harvesting. Cells were harvested by centrifugation, and native extracts were prepared and used to immunoprecipitate Slp1-HA (as above). Samples were immunoblotted with Anti-HA 12CA5 antibody to detect Slp1-HA and with anti-Ubiquitin antibody (Enzo, BML-PW8810) to detect ubiquitinated forms of Slp1-HA.

ALAL-1 siRNA knockdown was performed in HCC95 cells, and after 2 d, cells were lysed and immunoprecipitated with the appropriate antibody (anti-HA, mouse, sc-7392; anti-PRP3, rabbit, A302-074; or anti-TAK1, rabbit; Thermo Fisher; 700113) and protein G magnetic beads. IgG was used as a control for immunoprecipitation. 10% of the lysate was used as input. Ubiquitinated proteins were detected by immunoblot using a mono- and polyubiquitinylated conjugates monoclonal antibody (FK2, mouse; Enzo; #BML-PW8810).

Dynamin-like protein 1 (Dlp1/Drp1) (Clone 8) mouse antibody (BD Transduction Laboratories, BD611112, 1:1,000, 2% milk), mono- and polyubiquitinylated conjugated mouse monoclonal antibody clone FK2 (Enzo, BML-PW8810, 1:1,000, 5% milk), PINK1 rabbit polyclonal antibody (Novus Biologicals, BC100-494, 1:1,000, 2% BSA), Parkin (Prk8) mouse monoclonal antibody (Cell Signaling, 4211, 1:1,000, 2% milk), Rab5 (C8B1) rabbit monoclonal antibody (Cell Signaling, 3547, 1:1,000, 5% BSA), VDAC (D73D12) rabbit monoclonal antibody (Cell Signaling, 4661, 1:1,000, 2% BSA), GAPDH (14C10) rabbit monoclonal antibody (Cell Signaling, 2118, 1:10,000, 2% BSA), and LC3 A/B rabbit antibody (Cell Signaling, 4108, 1:1,000, 2% BSA).

The following primary antibodies were used in this study: goat anti-Galectin 8 (R&D Systems, AF1305; AB_2137229; 1:250 dilution), rabbit anti-CtsD (provided by Andrej Hasilik; 1:100 dilution), mouse anti-ubiquitin FK2 (Enzo, BML-PW8810; batch 08101527; AB_10541840; 1:100 dilution), rabbit anti-p62 (GeneTex, GTX111393; batch 40450; AB_10723101; 1:500 dilution), rabbit anti-NDP52 (provided by J. Kendrick-Jones; 1:250 dilution), rabbit andti-IRF-1 (Cell Signalling, 8478 lot 2; 1;250 dilution) and rabbit anti-p65 (Abcam, ab16502; batch GR3182233; 1:250 dilution). All were incubated for 1 h at RT at the dilution shown. The following secondary antibodies were used in this study at 1:800 dilution: goat anti-rabbit Alexa Fluor-568 (Thermo Fisher; A11036; Batch 1704462 AB_143011), goat anti-mouse Alexa Fluor-633 (Thermo Fisher; A21052; batch 1712097; AB_141459) and bovine anti-goat Cy3 (Jackson ImmunoResearch; 805-165-180; AB_2340880).

Flies were fixed for 20 min with 3.7% formaldehyde in PBS, and after fixation, hemi-thoraces were dissected. Samples were rinsed three times with 0.2% Triton X-100 in PBS (PBST) and blocked for 1 h at room temperature in 3% BSA in PBST. Primary antibodies (anti-FK2, mono- and polyubiquitinylated conjugates monoclonal antibody, BML-PW8810, Enzo) were diluted 1:250 in 5% BSA in PBST and samples were incubated overnight at 4 °C. Samples were rinsed thrice with PBST and incubated in a mix of 1:250 diluted secondary antibodies (anti-mouse AlexaFluor-488, Invitrogen) and a stain (phalloidin AlexaFlour-568, Invitrogen) in 5% BSA in PBST for 3 hours at room temperature. Samples were rinsed three times with PBST and mounted in Vectashield mounting medium (Vector Labs) and imaged using a Zeiss single point LSM 5 exciter confocal microscope. Protein aggregate and total muscle areas were quantified using ImageJ using the analyze particle features and the aggregate values were normalized by the area of muscle.

Proteins of the membrane-enriched fraction were loaded on acrylamide/bisacrylamide (29:1) gels and electrotransferred to nitrocellulose membranes, except for LC3 analysis, for which proteins were electrotransferred to polyvinylidene fluoride membranes. Antibodies against the following proteins were used for western blot analysis: Beclin-1 (Cell Signaling Technology, 3738), FYCO1 (Novus Biologicals, NBP1-47266), LAMP-2 (Abcam, ab13524), LC3 (Cell Signaling Technology, 2775), p62 (Sigma Aldrich, P0067), ribosomal protein S6 (Cell Signaling Technology, 2217), and ubiquitinated proteins (Enzo Life Sciences, BML-PW8810). Signals were revealed with the Clarity Western ECL substrate (Bio-Rad) and acquired with the ChemiDoc Touch Imaging System (Bio-Rad). Signals were quantified with the Image Lab Software (Bio-Rad).