To quantify protein expression, MeWo, MeWo-HS3ST4 (+), and MeWo-HS3ST4 (−) cells
at passage eight (P8) or P17 in 10 cm dishes were washed and suspended in
electrophoresis sample buffer (Santa Cruz Biotechnology), followed by
sonication. The samples were loaded onto e-PAGEL(R) 5–20% precast gels (ATTO).
The protein bands were transferred to a polyvinyl difluoride membrane
(Trans-Blot Turbo Midi 0.2 μm PVDF Transfer Packs, Bio-Rad) using a Trans-Blot
Turbo Transfer System (Bio-Rad), followed by blocking with Blocking One
(Nakalai) and blotting with anti-HS3ST4 sheep antibodies (R&D) and
anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1E6D9 mouse antibodies
(Proteintech) as primary antibodies and peroxidase donkey anti-sheep IgG (H+L)
and peroxidase donkey anti-mouse IgG (H+L) (Jackson Immunoresearch) as secondary
antibodies. The membrane was treated with horseradish peroxidase substrate
(WBKLS0100 Immobilon Western Chemiluminescent HRP Substrate, Merck).
Chemiluminescence was quantified using a Lumino Image Analyzer (ImageQuant
LAS-4000mini, Cytiva).
at passage eight (P8) or P17 in 10 cm dishes were washed and suspended in
electrophoresis sample buffer (Santa Cruz Biotechnology), followed by
sonication. The samples were loaded onto e-PAGEL(R) 5–20% precast gels (ATTO).
The protein bands were transferred to a polyvinyl difluoride membrane
(Trans-Blot Turbo Midi 0.2 μm PVDF Transfer Packs, Bio-Rad) using a Trans-Blot
Turbo Transfer System (Bio-Rad), followed by blocking with Blocking One
(Nakalai) and blotting with anti-HS3ST4 sheep antibodies (R&D) and
anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1E6D9 mouse antibodies
(Proteintech) as primary antibodies and peroxidase donkey anti-sheep IgG (H+L)
and peroxidase donkey anti-mouse IgG (H+L) (Jackson Immunoresearch) as secondary
antibodies. The membrane was treated with horseradish peroxidase substrate
(WBKLS0100 Immobilon Western Chemiluminescent HRP Substrate, Merck).
Chemiluminescence was quantified using a Lumino Image Analyzer (ImageQuant
LAS-4000mini, Cytiva).