Alexa fluor 546 protein labeling kit

RhGAA was kindly provided by Prof. Giancarlo Parenti. rhGAA (2 mg/ml) was conjugated with the Alexa Fluor™−546 Protein Labeling Kit (ThermoFisher, cat# A10237) following manufacturer instruction. Cells were incubated with saturating concentrations of rhGAA (1:50 dilution, 40 µg/ml) in complete media at 37 °C for 45 min, washed and chased for the indicated time points, and then fixed and processed for immunofluorescence. For live-image experiments, cells were loaded with 1 µg/µl dextran-488 for 1 h at 37 °C, and then extensively washed for 16 h in serum+glutamine starvation medium (prolonged starvation media). The next day, cells were loaded with 40 µg/ml rhGAA for 60 min at 37 °C and then washed prior to imaging.
Pepstatin A labels active cathepsin D inside lysosomes. For PepstatinA loading experiments, cells were loaded with 1 µM Pepstatin A-BODIPY® Conjugate (Life technologies, cat# P12271), for 30 min at 37 °C, and then fixed at 37 °C for 15 min via the addition of an equal volume of prewarmed, freshly made 8% PFA in 2× microtubule stabilization buffer (MTSB; 160 mM PIPES pH 6.8, 10 mM EGTA, 2 mM MgCl2) to minimize tubules rupture, as previously reported⁵⁹ .

Trastuzumab and bevacizumab (Roche, Genentech) were provided by the British Columbia Cancer Agency pharmacy; dilutions to 1–2 mg/ml were prepared in sterile 0.9% NaCl before intra-peritoneal (i.p.) injection. Human isotype control IgG1 (Sigma) was administered from similar concentrations. Trastuzumab and IgG antibodies for use in combination with bevacizumab were tagged with fluorescent labels according to Alexa Fluor 546 Protein Labeling Kit (ThermoFisher) instructions. Hypoxia marker pimonidazole (Hypoxyprobe) was administered at 60 mg/kg as an i.p. injection 2 h prior to tissue harvest. Fluorescent dye DiOC₇(3) (Molecular Probes), 0.6 mg/ml dissolved in 75% (v/v) dimethyl sulfoxide/25% sterile H₂O, was administered intravenously as a marker of vessel perfusion 5 min prior to tissue harvest [18 (link)].

Polyclonal anti-ROCK I (H-85) and anti-ROCK II (H-85) antibodies and monoclonal anti-Myc (9E10) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-α-actinin antibody and monoclonal anti-MLC pS19 antibody were purchased from Cell Signaling Technology (Danvers, MA). Monoclonal anti-MLC (MY-21) antibody, monoclonal anti-MLCK (K-36) antibody, gelatin, BDM, and ML-7 were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal anti-facsin antibody, Y27632, puromycin, and fibronectin were purchased from EMD Millipore (Billerica, MA). Alexa Fluor 488-phalloidin, the Alexa Fluor 546 protein labeling kit, Alexa Fluor 488- and Alexa Fluor 546-conjugated secondary antibodies, and Lipofectamine were purchased from Thermo Fisher Scientific (Waltham, MA). The horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The plasmid pCS2+ -GFP-UtrCH was purchased from Addgene (Cambridge, MA). The plasmid pEGFP-actin was purchased from Takara Bio USA, Inc. (Mountain View, CA). The plasmid pEGFP-N1-MLCK was described previously^{28 (link)}. The plasmids mCherry-UtrCH, pEF-Myc-ROCK II C.A. (a.a 6–553), and pEF-Myc-ROCK II K121G (a.a 6–553) were kindly provided by Dr. H. H. Lee (National Yang-Ming University, Taiwan).