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4 protocols using anti fosl1

1

Protein Expression Analysis of FOSL1 and FOSL2

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Cell culture pellets
were lysed using radioimmunoprecipitation assay buffer (Pierce, cat
no. 89901) that was supplemented with protease and phosphatase inhibitors
(Roche) and sonicated using a Bioruptor UCD-200 (Diagenode). Sonicated
lysates were centrifuged at 14,000 rpm for 30 min at 4 °C, and
supernatants were collected. Samples were estimated for protein concentration
(DC protein assay; Bio-Rad) and boiled in 6× Laemmli buffer (330
mM Tris-HCl, pH 6.8; 330 mM sodium dodecyl sulfate; 6% β-ME;
170 μM bromophenol blue; and 30% glycerol). Samples were then
loaded on gradient Mini-PROTEAN TGX precast protein gels (Bio-Rad)
and transferred to polyvinylidene fluoride membranes (Trans-Blot Turbo
transfer packs, Bio-Rad).
For protein expression analysis of
FOSL1 and FOSL2, the following Ab were used: anti-FOSL1 (Cell Signaling
Tech., cat no. 5281), anti-FOSL2 (Cell Signaling Tech., cat no. 19967),
and anti-β-actin (Sigma-Aldrich, cat no. A5441). HRP-conjugated
anti-mouse IgG (Santa Cruz Biotechnology, cat no. sc-2005) and anti-rabbit
IgG (BD PharMingen, cat no. 554021) were used as secondary Ab.
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2

Subcellular Fractionation of Th0 and Th17 Cells

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Cell pellets
of Th0 and Th17 cultures (24 and 72 h) were lysed and fractionated
into cytoplasmic and nuclear components using a NE-PER nuclear and
cytoplasmic extraction reagent kit (Thermo Fisher Scientific, cat
no. 78833) by following the manufacturer’s instructions. Extracts
were then analyzed by western blotting. FOSL localization was determined
using anti-FOSL1 (Cell Signaling Tech., cat no. 5281) and anti-FOSL2
(Cell Signaling Tech., cat no. 19967) Ab. Anti-GAPDH (HyTest, cat
no. 5G4) and anti-LSD1 (Diagenode, cat no. C15410067) Ab were used
to mark the cytoplasmic and nuclear fractions, respectively.
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3

Fractionation and Analysis of Th Cell Subsets

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Cell pellets of Th0 and Th17 cultures (24h and 72h) were lysed and fractionated into cytoplasmic and nuclear components using the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit, (Thermo Fischer Scientific, Cat no. 78833), by following the manufacturer's instructions. Extracts were then analysed by western blotting. FOSL localization was determined using anti-FOSL1 (Cell Signaling Tech, Cat no. 5281) and anti-FOSL2 (Cell Signaling Tech., Cat no.19967) antibodies. Anti-GAPDH (Hytest, Cat no. 5G4) and anti-LSD1 (Diagenode, Cat no. C15410067) antibodies were used to mark the cytoplasmic and nuclear fractions, respectively.
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4

Protein Expression Analysis of FOSL1 and FOSL2

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Cell culture pellets were lysed using RIPA buffer (Pierce, Cat no. 89901) that was supplemented with protease and phosphatase inhibitors (Roche) and sonicated using a Bioruptor UCD-200 (Diagenode). Sonicated lysates were centrifuged at 14,000 rpm for 30 min at 4°C, and supernatants were collected. Samples were estimated for protein concentration (DC Protein Assay; Bio-Rad) and boiled in 6x Laemmli buffer (330 mM Tris-HCl, pH 6.8; 330 mM SDS; 6% β-ME; 170 μM bromophenol blue; 30% glycerol). Samples were then loaded on gradient Mini-PROTEAN TGX Precast Protein Gels (BioRad) and transferred to PVDF membranes (Trans-Blot Turbo Transfer Packs, BioRad).
For protein expression analysis of FOSL1 and FOSL2, the following antibodies were used: anti-FOSL1 (Cell Signaling Tech, Cat no. 5281), anti-FOSL2 (Cell Signaling Tech., Cat no.19967) and anti-β-actin (SIGMA, Cat no. A5441). HRP-conjugated anti-mouse IgG (SantaCruz, Cat no. sc-2005) and anti-rabbit IgG (BD Pharmingen, Cat no. 554021) were used as secondary antibodies.
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