Laemmli s sample buffer

We assessed cleavage of PARP1 and Caspase-3 to confirm that cell death was mediated by apoptosis. OSCC cells infected with ING1 adenoviral constructs or adenoviral controls were washed with PBS, lysed in Laemmli's sample buffer (Bio-Rad, Mississauga, ON, Canada), sonicated on ice and aliquots containing 50μg of protein were loaded on 12.5% polyacrylamide gels and electrophoresis was performed at a constant 100V. Samples were transferred to nitrocellulose membrane (Whatman, Piscataway, NJ, USA) and probed with rabbit monoclonal anti-cleaved-caspase-3 (Cell Signaling Technology), mouse monoclonal anti-PARP1 and anti-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

20μg (for MFSD5) and 100μg (for MFSD11) from brain and kidney protein samples were mixed with Laemmlis Sample Buffer (Bio-Rad) and various concentrations of 2-mercaptoethanol (Fluka), and loaded onto 12% TGX Miniprotean gels (Bio-Rad) and allowed to run at 100V. A pre-stained molecular weight marker was used as reference (Thermo Fischer). The transfer was performed either by blotting onto PVDF membrane (Immobilon-P, Millipore) at 4°C at 50V for 60min or by using the Trans-Blot^® Turbo^™ Mini PVDF Transfer Packs and Trans-blot Turbo Transfer system (Bio-Rad), following the manufactures instructions. The membrane was then incubated in 5% milk (Blotting grade blocker, Bio-Rad) TTBS blocking solution for 60min. Western blot was run with the polyclonal primary antibodies anti-MFSD5 (1:500, goat, Santa Cruz, sc-243473) and anti-MFSD11 (1:100, rabbit, Sigma-Aldrich, HPA022001), as these antibodies were utilized for subsequent staining. The antibodies were added to the membrane and allowed to bind over night at 4°C. After 3x10min wash with TTBS the membrane was incubated at RT for 60min with HRP coupled secondary antibodies (anti-goat and anti-rabbit (Invitrogen), diluted 1:10000 in blocking solution). The membrane was developed using Clarity Western ECL Substrate (Bio-Rad) and visualized using a CCD camera (Bio-Rad), staining was compared to the molecular weight marker.

Proteins were extracted by using Ripa buffer (Thermo Fisher Scientific, Waltham, MA,USA) and concentrations were calculated with BCA assay (Thermo Scientific, Waltham, MA, USA) as previously reported [22 ]. Protein samples were diluted in Laemmli’s sample buffer (Biorad, CA, US), heated for 5 min at 95 ◦C, and separated by sodium dodecyl sulfate − polyacrylamide gel electrophoresis (SDS PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). PVDF membranes were probed with the primary (antiMyoD (MA1-41017; Thermo Scientific, Waltham, MA, USA), β ACTIN (BA3R; Thermo Scientific, Waltham, MA, USA); and secondary antibodies ( Anti-mouse (Cell Signaling, 7076). Signals were detected with a chemiluminescence reagent (ECL, Millipore, Burlington, MA, USA) and Images were recorded using a LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed as we previously reported [24 (link)].

Ripa buffer (Thermo Fisher Scientific, Waltham, MA,USA) was used to extract the protein and concentrations were calculated with BCA assay (Thermo Scientific, Waltham, MA, USA) as previously reported [14 (link)]. Protein samples were diluted in Laemmli’s sample buffer (Biorad, CA, US) and heated for 5 min at 95 °C, and then separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS PAGE). Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). PVDF membranes were probed with the primary (antiMyoD (MA1-41,017; Thermo Scientific, Waltham, MA, USA); β ACTIN (BA3R; Thermo Scientific, Waltham, MA, USA); sirtuin (PA5-23,063; Invitrogen, Waltham, MA, USA); SESN1 (PA5-98,142; Invitrogen, Waltham, MA, USA); SESN2 (ab-178518; Abcam, Cambridge, MA); p53 (2524; Cell Signaling Technology, Danvers, Massachusetts, USA); p21 (M7202; Dako, Denmark A/S); UCP1 (ab-10983; Abcam, Cambridge, MA)) and secondary antibodies Anti-mouse (7076; Cell Signaling Technology) and Anti-rabbit (7074, Cell Signaling Technology). Signals were detected with a chemiluminescence reagent (ECL, Millipore, Burlington, MA, USA) and images were recorded using a LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed as we previously reported [16 (link)].

3–5 μg of gdPT‐AF488 were mixed with Laemmli’s sample buffer (cat# 1610747, Biorad, Canada) and 2 mM dithiothreitol (DTT, cat# DTT‐RO, Millipore Sigma), and heated at 100°C for 5 min prior to electrophoresis in 16% tris‐glycine polyacrylamide gels for 1.5 h at 120 V. Gels were washed once with milliQ water for 10 min prior to in‐gel fluorescence using the PharosFX imaging system (Biorad). Data was analyzed using the Quantity One software. Afterwards, protein bands were stained in the same gels with InstantBlue™ (cat# ab119211, Abcam) overnight at RT. After 5 washes with milliQ water for 10 min each, protein bands were visualized using the GelDoc imaging system (Biorad). Data was analyzed using the Image Lab software.