Anti-FDFT1 antibody [EPR16481]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FDFT1 antibody [EPR16481] (AB195046)

Immunohistochemical analysis of paraffin-embedded human sebaceous carcinoma tissue sections labeling FDFT1 with ab195046 at a 1/16000 dilution. Goat anti-rabbit IgG H&L (HRP) ab97051 used as the secondary at a 1/500 dilution. Counterstain hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FDFT1 antibody [EPR16481] (AB195046)

Immunohistochemical analysis of paraffin-embedded human lung squamous carcinoma tissue sections labeling FDFT1 with ab195046 at a 1/16000 dilution. Goat anti-rabbit IgG H&L (HRP) ab97051 used as the secondary at a 1/500 dilution. Counterstain hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-FDFT1 antibody [EPR16481] (AB195046)

FDFT1 was immunoprecipitated from HepG2 whole cell extract with ab195046 at 1/70 dilution. Western blot was performed from the immunoprecipitate using ab195046 at 1/10000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1 : HepG2 whole cell extract (Input) 10 μg. Lane 2 : ab195046 IP in HepG2 whole cell extract. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab195046 in HeLa whole cell extract.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-FDFT1 antibody [EPR16481] (ab195046)

Predicted band size: 48 kDa

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• WB

Supplier Data

Western blot - Anti-FDFT1 antibody [EPR16481] (AB195046)

All lanes:

Western blot - Anti-FDFT1 antibody [EPR16481] (ab195046) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Rat kidney lysate at 10 µg

Lane 3:

Rat spleen lysate at 10 µg

Lane 4:

RAW264.7 lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa

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• WB

Supplier Data

Western blot - Anti-FDFT1 antibody [EPR16481] (AB195046)

All lanes:

Western blot - Anti-FDFT1 antibody [EPR16481] (ab195046) at 1/10000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 48 kDa

false

• WB

Supplier Data

Western blot - Anti-FDFT1 antibody [EPR16481] (AB195046)

All lanes:

Western blot - Anti-FDFT1 antibody [EPR16481] (ab195046) at 1/10000 dilution

All lanes:

HepG2 cell lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 48 kDa

false

• WB

CiteAb

Western blot - Anti-FDFT1 antibody [EPR16481] (AB195046)

Western Blotting using Anti-FDFT1 antibody [EPR16481], ab195046. Publication image from Weng, M. L. et al., 2020, Nat Commun, 32313017. Legend direct from paper.

Fasting upregulates the level of FDFT1, which is correlated with prognosis in CRC.a The expression of FDFT1 was increased significantly in the fasting group compared with that in the control group in the GSE60653 data set (n = 3). b The relative expression of FDFT1 was also increased greatly in the fasting group compared with that in the control group by iTRAQ (n = 3; P = 0.0319). c The mRNA expression of FDFT1 in dissected tumor tissue from the fasting mimic group and the control group was measured by qRT-PCR (n = 15; P < 0.0001). d, e Fasting mimic medium also increased the protein level of FDFT1 in CT26 and SW620 cells. f Representative graph of the IHC analysis carried out in human CRC and noncancerous tissues (n = 23; upper : scale bar is 200 µm; lower : scale bar is 100 µm). g The expression of FDFT1 was downregulated in most of the tumor tissues (19/23), but was upregulated in most of the adjacent noncancerous tissues (18/23) (n = 23; P = 0.0004). h The relative expression levels of FDFT1 mRNA in CRC tissues and matched adjacent noncancerous tissues were determined by qRT-PCR (n = 81; both P < 0.0001). i Kaplan–Meier analysis of the overall survival of patients with CRC in the FUSCC cohort according to FDFT1 expression. The median expression level was used as the cutoff. High FDFT1 expression predicted better prognoses for CRC patients in the FUSCC cohort. (high FDFT1 patients = 39, low FDFT1 patients = 42; P = 0.0238, log-rank test) j, k The expression of the FDFT1 gene was significantly lower in CRC tissues than in normal tissues in the GDS2609 and GDS4382 data sets (P < 0.0001; P = 0.0049). l Analysis of the correlation of FDFT1 expression with TNM stage in CRC patients. Lower FDFT1 expression was correlated with higher TNM stage (P = 1.91 x 10−5). m Survival analysis of FDFT1 data from the TCGA database stratified by FDFT1 expression. High FDFT1 expression indicated a better prognosis. (P = 0.018, log-rank test). Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. Box denotes 25th to 75th percentile, horizontal bar is median in h, j, and k. Kaplan–Meier analysis and log-rank tests were used in panels i, m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group (or non-tumor/normal tissue).

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• WB

CiteAb

Western blot - Anti-FDFT1 antibody [EPR16481] (AB195046)

Western Blotting using Anti-FDFT1 antibody [EPR16481], ab195046. Publication image from Weng, M. L. et al., 2020, Nat Commun, 32313017. Legend direct from paper.

FDFT1 is a downstream target of fasting in suppressing CRC proliferation.aFDFT1 overexpression and fasting for 48 h inhibited CT26 cell proliferation as measured by a CCK8 assay. Compared with either treatment alone, FDFT1 overexpression combined with fasting for 48 h had the most obvious inhibitory effect on CT26 cell proliferation (from left to right : P = 0.0021; P = 0.0005; P = 0.0003). b Western blotting indicated that fasting 48 h and FDFT1 overexpression increased the protein level of FDFT1 in CRC cells. Fasting exerted an additive effect on FDFT1 expression level in cells overexpressing FDFT1 in the suppression of CRC cell proliferation. c, d Photograph of dissected tumors (first line : CT26 cells + normal diet; second line : CT26 cells + FMD; third line : FDFT1-overexpressing CT26 cells + normal diet; fourth line : FDFT1-overexpressing CT26 cells + FMD; n = 4; both P < 0.0001). Both the FMD and FDFT1 overexpression inhibited tumor growth in the mice. The FMD combined with the implantation of FDFT1-overexpressing CT26 cells had the most obvious inhibitory effect on tumor growth in the mice. e Representative 18F-FDG microPET/CT imaging of tumor-bearing mice. f The ratio of the tumor SUVmax in the four groups. The SUVmax was decreased most significantly in the FDFT1-overexpressing CT26 cells + FMD group (n = 3; from left to right : P = 0.0018; P = 0.0018; P = 0.0003). g The protein expression of FDFT1 in dissected tumor samples was evaluated by IHC. Scale bar : 100 µm. h Graph shows the quantitative analysis of FDFT1 staining (n = 3). i The effect of FDFT1 knockdown, fasting 48 h and FDFT1 knockdown combined with fasting 48 h on CT26 cell proliferation was evaluated by CCK8 (upper : P = 0.005; lower : P = 0.0045). j, k Photograph of dissected tumors (first line : CT26 cells + normal diet; second line : CT26 cells + FMD; third line : shFDFT1 CT26 cells + normal diet; fourth line : shFDFT1 CT26 cells + FMD; n = 4; P = 0.0006; P = 0.0002; P = 0.0003). The FMD inhibited tumor growth in mice. FDFT1 knockdown promoted tumor growth in mice. The FMD combined with shFDFT1 CT26 cells did not inhibit tumor growth in the mice. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group (or normal diet group). #P < 0.05, ##P < 0.01.

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• WB

CiteAb

Western blot - Anti-FDFT1 antibody [EPR16481] (AB195046)

Western Blotting using Anti-FDFT1 antibody [EPR16481], ab195046. Publication image from Weng, M. L. et al., 2020, Nat Commun, 32313017. Legend direct from paper.

Fasting and mTOR inhibitor synergize in suppressing CRC proliferation and clinical significance of the FDFT1/AKT-mTOR-HIF1α pathway in CRC patients.a CT26 cells were injected into BALB/c mice. When the tumors were palpable, the mice were randomly assigned to the normal diet group, FMD group, the rapamycin 1 mg/kg group and FMD + rapamycin 1 mg/kg group. Photograph of dissected tumors (the first line : normal diet, the second line : FMD, the third line : rapamycin 1 mg/kg, the fourth line : FMD + rapamycin 1 mg/kg, n = 5). b The tumor volumes were measured every 3 days after the 9th day (n = 30; ***P = 0.0008, P = 0.0003; #P = 0.0133). On day 9 after inoculation, all the tumor were palpable. c Kaplan–Meier analysis of the overall survival of mice after the inoculation in normal diet group, FMD group, normal diet mice treated with rapamycin 1 mg/kg group and FMD + rapamycin 1 mg/kg group (n = 30; log-rank score : P = 0.0049 for FMD group, P = 0.0058 for rapamycin 1 mg/kg group; P = 0.00069 for FMD + rapamycin 1 mg/kg group.) d, e The expression level of FDFT1 in four groups was evaluated by western blotting and qRT-PCR (**P = 0.0025, P = 0.0097; ***P = 0.0008; #P = 0.0133, P = 0.0351). f–j Survival analysis stratified by combining FDFT1 levels with AKT1, mTOR, HIF1α, GLUT1, and HK2 levels from CRC patients in the TCGA cohort. k Proposed model of the mechanism underlying the fasting-mediated regulation of glucose metabolism via the FDFT1/AKT-mTOR-HIF1α axis in colorectal cancer. Fasting upregulates the expression of FDFT1 during the inhibition of colorectal cancer cell aerobic glycolysis and proliferation. FDFT1, whose downregulation is correlated with malignant progression and poor prognosis in CRC, acts as a critical tumor suppressor in CRC. We then observed that FDFT1 is an important downstream target of fasting that mediates the inhibition of CRC cell proliferation. Mechanistically, FDFT1 inhibits the AKT-mTOR-HIF1α pathway, impairing aerobic glycolysis, and thereby suppressing the proliferation of CRC cells. There is also a reverse regulation of FDFT1 by mTOR. Error bars, mean ± SD, the data are from three independent experiments. Two-sided t tests. Kaplan–Meier analysis and log-rank tests were used in panel c. *P < 0.05, **P < 0.01, ***P < 0.001, compared with normal diet group. #P < 0.05, ##P < 0.01.

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