Ab141446

For pharmacological inhibition, cells were treated using the following concentration of drugs prior to sample fixation or laser ablation experiment: sodium orthovanadate (S6508, Sigma) 100 μM, RK682 (RK2033, Sigma) 10 μg/ml; ALLN (ab141445, Abcam) 10 and 50 μM, ALLM (ab141446, Abcam) 10 and 50 μM, nocodazole (M1404, Sigma) 10 μM, and Y-27632 (Y0503, Sigma) 10 μM.
While sodium orthovanadate is a generic alkaline and tyrosine phosphatase inhibitor, RK682 is a specific and non-competitive inhibitor of protein tyrosine phosphatase with confirmed low micromolar inhibitory activity against protein tyrosine phosphatases CD45 and PTP1B, and dual-specificity phosphatases VHR, CDC-25A-B-C (Hamaguchi et al., 1995 (link)); nevertheless, CD45 and VHR genes are not expressed in MDCK and EpH4 cells, as indicated by RNAseq data (Supplementary Table 1), while inhibition of CD25 requires longer incubation period (20 h) with respect to what we have used in the current paper (1 h).
ALLN and ALLM are inhibitors for calpains, with high binding efficiency for calpain-1 and calpain-2, respectively. ALLN has been reported to inhibit the calpain/PTP1B axis in endothelial cells (Zhang et al., 2017 (link)).

Cathepsin B activity was assayed in the cortex of 3 or 6-month-old mice using a fluorometric kit (Abcam, #ab65300). Tissue was homogenised in lysis buffer then incubated for 30 minutes on ice, then prior to centrifugation at 15,000 x g for 5 minutes at 4°C. The supernatant was removed and transferred to a clean tube, on ice, and the protein concentration was determined by a Bradford assay. For each sample, a volume of supernatant containing 200μg protein used for each reaction with cathepsin B Substrate (RR-amino-4-trifluoromethyl coumarin (AFC)). To control for non-specific cleavage, negative controls treated with inhibitor ALLM (Abcam, ab141446), were run for every sample. Recombinant human cathepsin B (R&D Systems, Cat. No. 953-CY-010) was used as a positive control (0.01 μg for each control reaction). The reaction was incubated at 37°C and the fluorescent output (excitation/emission = 400/505nm) was recorded every 90 seconds for 30 cycles. The linear range of the reaction was determined and the relative cathepsin B activity in the sample calculated by taking the average fluorescent output for each sample minus that in the inhibited. Values are expressed as a percentage of WT average.