Pretreated cells were gathered on ice and lysed with RIPA lysis buffer. Proteins were separated on 4%–12% Bis-Tris polyacrylamide gels and transferred to the PVDF membrane. The blots were incubated with primary antibodies overnight at 4°C and then with secondary antibodies. The following primary antibodies were adopted: Actin (Servicebio, GB12001; 1 : 1000); Aldoc (Proteintech, 14884-1-AP, 1 : 5000); CD74 (Bioss, bs-2518R, 1 : 2000); CYP51 (Proteintech, 13431-1-AP, 1 : 5000); EBP (Proteintech, 15518-1-AP, 1 : 1000); ENO2 (Servicebio, GB11376, 1 : 1000); FDFT1 (Proteintech, 13128-1-AP, 1 : 1000); FOXO1 (Servicebio, GB11286; 1: 2000); Gapdh (Proteintech, 60004-1-Ig, 1 : 20000); Gpi1 (Proteintech, 15171-1-AP, 1 : 1000); Hif-1a (Servicebio, GB111339, 1 : 1000); Mif (Proteintech, 26992-1-AP, 1 : 1000); NSDHL (Proteintech, 15111-1-AP, 1 : 1000); PFKL (abcam, ab181064, 1 : 5000); Pgk1 (abcam, ab199438, 1 : 2000); PKM (abcam, ab150377, 1 : 5000); p53 (Proteintech, 10442-1-AP, 1 : 1000); Tpi1 (abcam, ab196618, 1 : 1000).
Fdft1
Manufactured by Proteintech
FDFT1 is an enzyme involved in the biosynthesis of cholesterol. It catalyzes the conversion of farnesyl diphosphate to squalene, which is a key step in the cholesterol synthesis pathway. The FDFT1 gene encodes the squalene synthase enzyme responsible for this reaction.
Automatically generated - may contain errors
Lab products found in correlation
Ab199438, by Abcam (1 mentions)
Ab181064, by Abcam (1 mentions)
Aldoc, by Proteintech (1 mentions)
General sp kit, by ZSGB-BIO (1 mentions)
Dab substrate, by ZSGB-BIO (1 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
β actin, by ABclonal (1 mentions)
Irdye 680rd donkey anti rabbit, by LI COR (1 mentions)
Bp 115, by Boston BioProducts (1 mentions)
Irdye 800cw donkey anti mouse, by LI COR (1 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
4 protocols using fdft1
1
Comprehensive Western Blot Analysis of Metabolic Regulators
2
Immunohistochemical Analysis of FDFT1 and Ki-67 in Tissue Microarray
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A total of 450 samples were fixed by formalin, embedded in paraffin, and used for TMA construction. Tissue slides with paraffin sections were deparaffinized by xylene and ethanol and used for IHC analyses. Endogenous peroxidase activity was blocked with 3% H2O2 for 15 minutes at room temperature. For antigen retrieval, slides were heated in citrate buffer for 20 minutes. Nonspecific binding was blocked with 10% FBS in PBS for 15 minutes at 37°C. The slides were incubated with FDFT1 (1:200 dilution; Cat# 13128‐1‐AP; Proteintech), Ki‐67 (1:5000 dilution; Cat# ab15580; Abcam) at 4°C overnight. The slides were incubated with biotin‐conjugated secondary Ab using a general SP kit (Cat# SP‐9000; ZSGB‐BIO) and stained with the DAB substrate (Cat# SP‐9000; ZSGB‐BIO). The immunoreactivity of tested samples was scored for multiplying staining intensity (0‐3) and positive cell proportion (1‐4).
3
Western Blot Analysis of Lipid Metabolism Proteins
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Cells were washed with PBS and RIPA buffer (Cat# P0013B; Beyotime), PMSF (ST506; Beyotime), and phosphatase inhibitor cocktail (Cat#P1050; Beyotime) were added. Total proteins were separated by SDS‐PAGE and then transferred onto PVDF membranes (Cat# IPVH00010; Millipore). Membranes were incubated with primary Abs against FDFT1 (1:1000 dilution; Cat# 13128‐1‐AP; Proteintech), NAT8 (1:500 dilution; Cat# A7759; Abclonal), SQLE (1:1000 dilution; Cat# 12544‐1‐AP; Proteintech), Tubulin (1:5000 dilution; Cat# E7; DSHB), and β‐actin (1:5000 dilution; Cat# AC026; Abclonal) after being blocked at 4°C. Proteins were visualized using an ECL system following incubation with the secondary Abs against Goat anti‐Rabbit IgG (H + L) Cross‐Adsorbed Secondary Antibody, HRP (Cat# G21234; Invitrogen), and Goat anti‐Mouse IgG (H + L) Cross‐Adsorbed Secondary Antibody, HRP (Cat# G21040; Invitrogen).
4
Immunoblotting Analysis of FDFT1 Protein Expression
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Cells were washed once with 1× phosphate-buffered saline (PBS) and harvested in 1× RIPA buffer (BP-115, Boston Bioproducts) containing phosphatase and protease inhibitor cocktail (5872S, Cell Signaling Technologies). Cell lysates were briefly sonicated and subsequently cleared by centrifugation at 14K rpm for 10 min at 4 °C. Protein quantification was done by Pierce BCA Assay (23225, Life Technologies). For immunoblotting analysis, lysates were loaded onto precast sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (5671093, Bio-Rad) and subsequently transferred onto nitrocellulose membrane for detection. All primary antibodies were probed overnight at 4 °C, and membranes were washed with TBST and incubated with appropriate secondary antibodies for 1 h. Subsequently membranes were washed with TBST and visualized using Odyssey imaging system (LI-COR).
Primary antibodies used were FDFT1 (13128–1-AP, Proteintech, 1:1000), and β-actin (3700S, Cell Signaling, 1:5000). Secondary antibodies used were IRDye 680RD Donkey anti-Rabbit (926–68073, LI-COR, 1:5000) and IRDye 800CW Donkey anti-Mouse (926–32212, LI-COR, 1:5000).
Uncropped images are provided as Supplementary Fig.9 .
Primary antibodies used were FDFT1 (13128–1-AP, Proteintech, 1:1000), and β-actin (3700S, Cell Signaling, 1:5000). Secondary antibodies used were IRDye 680RD Donkey anti-Rabbit (926–68073, LI-COR, 1:5000) and IRDye 800CW Donkey anti-Mouse (926–32212, LI-COR, 1:5000).
Uncropped images are provided as Supplementary Fig.
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