Nb200 11

Proteins were separated on SDS-PAGE (8-12%) gels and
transferred to PVDF membranes (Millipore, Germany).
Membranes were blocked with 5% non-fat milk in PBS0.1%
TweenTM20 and were incubated overnight at 4°C
with the respective specific primary antibody for each
protein [mouse anti-LRSAM1/Abcam ab73113 (1:400),
rabbit anti-LRSAM1/Novus Biological H00090678-D01
(1:750), rabbit anti-CASPASE-3/Santa Cruz SC7148
(1:700), mouse anti-CYCLIN D1/Abcam ab6152 (1:300),
mouse anti-TSG101/Novus Biological NB200-11 (1:500)
and mouse anti-ß-ACTIN/Sigma-Aldrich A2228 (1:4000)]
which was diluted in phosphate buffered saline (PBS)-0.1%
TweenTM20. The membranes were then incubated for 2 hours
with the appropriate secondary antibodies [AP124P goat
anti-mouse IgG-Peroxidase H+L/Millipore (1:7000) and
sc-2077 donkey anti-rabbit IgG-HRP/ Santa Cruz (1:7000)]
followed by incubation with the visualization LumiSensorTM
Chemiluminescent HRP Substrate Kit (Genscript, USA).
Membranes were finally visualized using the UVP imaging
system (BioRad, USA). Western blots were quantified using
the ImageJ software (https://imagej.nih.gov). Protein quantity
ratio was estimated relative to ß-actin.

Proteins from each sample were loaded and separated onto SDS-PAGE 12% polyacrylamide gels and were transferred to Hybond-P hydrophobic polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). Membranes were blocked for 1 hour at room temperature and were incubated overnight at 4°C with the respective specific primary antibody for each protein [mouse anti-LRSAM1/Abcam ab73113 (1:400), rabbit anti-LRSAM1/Novus Biological H00090678-D01 (1:750), mouse anti-TSG101/Novus Biological NB200-11 (1:500) and mouse anti-β-ACTIN/Sigma-Aldrich A2228 (1:4000)]. Then, incubation with the appropriate secondary antibodies was performed for 2 hours and followed by incubation with the visualization LumiSensor Chemiluminescent HRP Substrate Kit (Genscript, U.S.A.) at room temperature. Membranes were visualized using the UVP imaging system (BioRad, U.S.A.). Quantification of Western blots was performed using the ImageJ program (https://imagej.nih.gov). The protein quantity ratio was estimated relative to β-Actin.