Nonidet p 40

Cells were lysed and assayed as previously reported [10 (link)]. Briefly, cells were lysed in cytoplasm lysis buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.2 mM EDTA, 1 mM DTT), containing protease inhibitors, 0.5 mM phenylmethylsulfonylfluoride (PMSF) and 0.6% Nonidet P-40 (Sigma Chemical Co., St Louis, MO, USA). Lysates were centrifuged at 10.000 rpm 10 min at 4°C and the supernatants (cytoplasmic fractions) were split into aliquots and rapidly frozen. The nuclear pellet was washed in buffer A without Nonidet P-40 and finally resuspended in nuclear lysis buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 2 mM EDTA, 1 mM DTT), containing protease inhibitors and 1 mM PMSF (Sigma Chemical Co., St Louis, MO, USA). Samples were incubated on ice 30 min and centrifuged at 13.000 rpm 10 min at 4°C; the supernatants (nuclear fractions) were split into aliquots and rapidly frozen or used for western blot analysis.

The probe for the (GATA)_n microsatellite motif was synthesized and biotin-labelled by Macrogen (Korea). The probe was resuspended in hybridization buffer (50% formamide, 10% sodium dodecyl sulphate (SDS), 10% dextran sulphate, Denhard’s buffer, 2× SSC, pH 7). We followed the same protocol used for FISH with the telomeric probe, except that the post-hybridization washes were performed in 0.4% Nonidet P-40 in 2× SSC (Sigma-Aldrich) at 40 °C for 2 min and then in 0.1% Nonidet P-40 in 0.4× SSC at room temperature for 30s, instead of 50% formamide in 2× SSC.

SK-BR-3 cells were treated with 5, 100, and 500 nM of Z_HER2:2891DCS-MMAE and incubated at 37 °C for 24, 48, and 96 h, while MDA-MB-231 cells were treated with 5, 100, and 500 nM of Z_HER2:2891DCS-MMAE and incubated for 24 h.
For the preparation of total cell lysates, cells were washed with ice-cold PBS and lysed with lysis buffer (NaCl 150 mM, TRIS 50 mM pH 7.6, NONIDET P-40 0.5% and protease inhibitors (Merck, Milan, Italy)).
Protein concentration was determined using a Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA) and samples were run on SDS-PAGE.
HER2 expression was evaluated by using a primary ErbB2 (HER2) monoclonal antibody (3B5) (Thermo Fisher Scientific) diluted 1:1000.
Densitometric analysis was performed using the ImageJ program.

Generally, cells were washed twice in ice-cold PBS then scraped in lysis buffer (50 mM Tris-HCl (pH 7.4), 270 mM sucrose, 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 1 mM sodium orthovanadate, 10 mM sodium β-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, and 1% (v/v) Nonidet P40 (492016, Merck) supplemented with 1× EDTA-free protease inhibitor cocktail (11873580001, Merck). Lysates were incubated on ice for 10 min, then clarified by centrifugation at 17,000g for 10 min at 4 °C. The supernatant (soluble cell extract) was collected in a fresh Eppendorf and either snap frozen in liquid nitrogen and stored at −80 °C for later processing or processed immediately after the protein concentration was measured in a 96-well format using the Bradford protein assay reagent (23236, Pierce).
TurboID experiments were adapted from the method described by Branon et al. [35 (link)]. Briefly, Cells were incubated with biotin (B4501, Sigma Aldrich) for 10 min at 37 °C then washed five times in ice cold PBS. Cells were then scraped in PBS, pelleted by centrifugation at 300g for 3 min at 4 °C, lysed in RIPA lysis buffer supplemented with 1× EDTA-free protease inhibitor cocktail (11873580001, Merck), then clarified as described above.

For the preparation of total cell lysates, cells were washed with ice-cold PBS and lysed with lysis buffer (NaCl 150 mM, TRIS 50 mM pH 7.6, NONIDET P-40 0.5%, and protease inhibitors (Merck, Milan, Italy)). Protein concentration was determined using a Pierce BCA Protein Assay kit (Pierce, Rockford, IL, USA) and samples were run on SDS-PAGE. The different proteins were detected using specific primary antibodies: ACTA-2 (ab7817, 1:300), LGALS-3 (ab76245, 1:5000), and IL-1β (ab2105, 1:200) were from Abcam (Cambridge, UK); tubulin (T6199, 1:1500) from Sigma-Aldrich (Milan, Italy). Quantification was performed using densitometric analysis using Image Studio Lite software v. 3.1 from Li-Cor Bioscience (Lincoln, NE, USA).