The ER stress markers were identified
by Western blot analysis, using a Wes Simple Western Blot device (ProteinSimple).
Cells were seeded at 3 × 105 cells per well in a 6-well
plate and incubated for 2–3 days. Cells were then treated with
cubic ND-PEG-SPIONs (0.100 mg/mL Fe) for 24 h. After replacing particle-containing
media with fresh media, cells were treated with pulsed magnetic field
and then incubated for 3 h at 37 °C, 5% CO2. Cells
were abundantly washed with PBS, trypsinized, and centrifuged, and
then 100 μL of RIPA lysis buffer 10× (EMD Millipore; RIPA
containing protease inhibitor cocktail from cOmplete) was applied
to each well for 0.5 h. Then, 30 uL (out of 100 uL of lysate, 70 uL
was used for next step) of lysate diluted 3× using DPBS was used
for BCA analysis as described above to determine protein concentrations.
Using BCA data, 250 μg/mL of protein was denatured and loaded
in Wes multiwell plates following the manufacturer’s protocol.
The protein bands were detected with primary antibodies described
inTable S2 (Supporting Information) and
secondary Goat Anti-Rabbit HRP Conjugate (ready-to-use reagent, ProteinSimple).78 (link)
by Western blot analysis, using a Wes Simple Western Blot device (ProteinSimple).
Cells were seeded at 3 × 105 cells per well in a 6-well
plate and incubated for 2–3 days. Cells were then treated with
cubic ND-PEG-SPIONs (0.100 mg/mL Fe) for 24 h. After replacing particle-containing
media with fresh media, cells were treated with pulsed magnetic field
and then incubated for 3 h at 37 °C, 5% CO2. Cells
were abundantly washed with PBS, trypsinized, and centrifuged, and
then 100 μL of RIPA lysis buffer 10× (EMD Millipore; RIPA
containing protease inhibitor cocktail from cOmplete) was applied
to each well for 0.5 h. Then, 30 uL (out of 100 uL of lysate, 70 uL
was used for next step) of lysate diluted 3× using DPBS was used
for BCA analysis as described above to determine protein concentrations.
Using BCA data, 250 μg/mL of protein was denatured and loaded
in Wes multiwell plates following the manufacturer’s protocol.
The protein bands were detected with primary antibodies described
in
secondary Goat Anti-Rabbit HRP Conjugate (ready-to-use reagent, ProteinSimple).78 (link)
