Anti-PDPK1 antibody [EP569Y]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDPK1 antibody [EP569Y] (AB52893)

ab52893 (1 : 100) staining human PDPK1 in human prostate carcinoma tissue by immunohistochemistry using paraffin embedded tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDPK1 antibody [EP569Y] (AB52893)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling PDPK1 with Purified ab52893 at 1 : 150 dilution(2.4 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Purified)ImmunoHistoProbe one step HRP Polymer (ready to use) was used for detection. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Supplier Data

Western blot - Anti-PDPK1 antibody [EP569Y] (AB52893)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : PDPK1 knockout HAP1 cell lysate (20 μg)
Lane 3 : HEK293 cell lysate (20 μg)
Lane 4 : MCF7 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab52893 observed at 70 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab52893 was shown to recognize PDPK1 when PDPK1 knockout samples were used, along with additional cross-reactive bands. Wild-type and PDPK1 knockout samples were subjected to SDS-PAGE. ab52893 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PDPK1 antibody [EP569Y] (ab52893)

Predicted band size: 63 kDa

false

• WB

Lab

Western blot - Anti-PDPK1 antibody [EP569Y] (AB52893)

The molecular weight observed is consistent with what has been described in PMID : 30171166

All lanes:

Western blot - Anti-PDPK1 antibody [EP569Y] (ab52893) at 1/1000 dilution

Lane 1:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Lane 2:

Rat testis lysates at 20 µg

Lane 3:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates at 20 µg

Lane 4:

22Rv1 (Human prostate carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 5:

PIEC (PIG iliac artery endothelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 63 kDa

Observed band size: 52-65 kDa

false

• WB

Lab

Western blot - Anti-PDPK1 antibody [EP569Y] (AB52893)

The molecular weight observed is consistent with what has been described in PMID : 30171166

All lanes:

Western blot - Anti-PDPK1 antibody [EP569Y] (ab52893) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/5000 dilution

Predicted band size: 63 kDa

Observed band size: 52-65 kDa

false

• WB

Lab

Western blot - Anti-PDPK1 antibody [EP569Y] (AB52893)

The molecular weight observed is consistent with what has been described in PMID : 30171166

All lanes:

Western blot - Anti-PDPK1 antibody [EP569Y] (ab52893) at 1/1000 dilution

All lanes:

F9 (Mouse embryonal carcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/5000 dilution

Predicted band size: 63 kDa

Observed band size: 52-65 kDa

false

• WB

Unknown

Western blot - Anti-PDPK1 antibody [EP569Y] (AB52893)

All lanes:

Western blot - Anti-PDPK1 antibody [EP569Y] (ab52893) at 1/1000 dilution

All lanes:

293 cell lysate

Predicted band size: 63 kDa

Observed band size: 63 kDa

false

• WB

CiteAb

Western blot - Anti-PDPK1 antibody [EP569Y] (AB52893)

Western Blotting using Anti-PDPK1 antibody [EP569Y], ab52893. Publication image from Oh, J. et al., 2023, Nat Commun, 37311757. Legend direct from paper.

Macrophage miR-106b-5p mediates in utero VD deficiency-induced adipocyte IR.A–C Quantitative RT-PCR, Western blot analysis, and densitometry (normalized to β-actin protein levels) of the insulin signaling pathway in 3T3-L1 cells after transfection with miR-106b-5p mimic vs. control mimic (n = 4/group). D–F Quantitative RT-PCR, western blot analysis, and densitometry (normalized to β-actin protein levels) of the insulin signaling pathway in 3T3-L1 cells cultured in conditioned media from VD(−) HSC-recipient macrophages after transfection with anti-miR-106b or control (n = 4/group) from two independent experiments. G miR-106b-5p expression in adipocytes cultured in conditioned media from macrophages isolated from VD(−) or VD( + ) HSC recipients (n = 4/group). H Pre- and mature miR-106b-5p abundance in 3T3-L1 adipocytes transfected with pre-miR-106b siRNA vs. control-siRNA then cultured in conditioned media from macrophages isolated from VD(−) or VD( + ) HSC recipients (n = 4/group). Peritoneal macrophage media miR-106b-5p expression from I VD(−) HSC-recipient macrophages with or without Ppargc1a-siRNA, and J VD( + ) HSC-recipient macrophages with or without Jarid2-siRNA (n = 6/group). K–M Fetal HSCs from WT or miR-106b−/− animals under VD(−) or VD(+) conditions were transplanted into VD( + ) WT recipients. K Glucose tolerance tests and L insulin tolerance tests (n = 8/group). M Insulin-stimulated 2-DG uptake in 3T3-L1 adipocytes co-cultured with peritoneal macrophages from WT or miR-106b−/− animals transplanted with VD(−) or VD(+) HSCs (n = 6/group). Data presented as mean ± SEM. A, C, D, F, H, I, J *P < 0.05; ***P < 0.001 by two-tailed unpaired t test. G, M *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA followed by Tukey’s multiple comparison test. K, L *P < 0.05; **P < 0.01; ***P < 0.001 VD(−) WT vs. all and †P < 0.05; †††P < 0.001 for VD( + ) WT vs. VD(−) miR-106b−/−. Actual P values shown in source data file.

false

• WB

CiteAb

Western blot - Anti-PDPK1 antibody [EP569Y] (AB52893)

Western Blotting using Anti-PDPK1 antibody [EP569Y], ab52893. Publication image from Oh, J. et al., 2023, Nat Commun, 37311757. Legend direct from paper.

VD-deficient cord blood monocytes induce adipocyte IR.A Cord blood serum 25(OH)D levels from 30 healthy pregnant women at delivery. Mean and 95% confidence interval. B Correlation between cord blood serum 25(OH)D levels and birth weight using Spearman’s correlation coefficient. C Correlation between cord blood serum 25(OH)D levels and change in insulin-stimulated 2-DG uptake in 3T3-L1 adipocytes cultured in conditioned media of cord blood monocytes using Spearman’s correlation coefficient. Western blot analysis of insulin signaling pathway from 3T3-L1 adipocytes exposed to cord blood monocytes (D) and from Jarid2/Mef2/PGC1α network-related proteins of cord blood monocytes (E) (n = 4/group from two independent experiments). F Quantitative RT-PCR of mRNA expression of Jarid2/Mef2/PGC1α network-related genes from cord blood monocytes stratified by 25(OH)D level (25(OH)D < 20 ng/mL and ≥20 ng/mL) (n = 4/group). G Correlation between cord blood serum 25(OH)D level and serum miR-106b-5p expression (n = 30) using Spearman’s correlation coefficient. H Insulin-stimulated 2-DG uptake in 3T3-L1 adipocytes transfected with miR-106b-5p antagomir and cultured in conditioned media from blood monocytes with 25(OH)D < 20 ng/mL or ≥20 ng/mL (n = 6/group). I Mechanistic schematic diagram Created with BioRender.com. Data presented as mean ± SEM. F *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed unpaired t test. H **P < 0.01; ***P < 0.001 by one-way ANOVA followed by Tukey’s multiple comparison test. Actual P values are shown in the source data file.

false

• WB

CiteAb

Western blot - Anti-PDPK1 antibody [EP569Y] (AB52893)

Western Blotting using Anti-PDPK1 antibody [EP569Y], ab52893. Publication image from Oh, J. et al., 2023, Nat Commun, 37311757. Legend direct from paper.

Macrophage miR-106b-5p mediates in utero VD deficiency-induced adipocyte IR.A–C Quantitative RT-PCR, Western blot analysis, and densitometry (normalized to β-actin protein levels) of the insulin signaling pathway in 3T3-L1 cells after transfection with miR-106b-5p mimic vs. control mimic (n = 4/group). D–F Quantitative RT-PCR, western blot analysis, and densitometry (normalized to β-actin protein levels) of the insulin signaling pathway in 3T3-L1 cells cultured in conditioned media from VD(−) HSC-recipient macrophages after transfection with anti-miR-106b or control (n = 4/group) from two independent experiments. G miR-106b-5p expression in adipocytes cultured in conditioned media from macrophages isolated from VD(−) or VD( + ) HSC recipients (n = 4/group). H Pre- and mature miR-106b-5p abundance in 3T3-L1 adipocytes transfected with pre-miR-106b siRNA vs. control-siRNA then cultured in conditioned media from macrophages isolated from VD(−) or VD( + ) HSC recipients (n = 4/group). Peritoneal macrophage media miR-106b-5p expression from I VD(−) HSC-recipient macrophages with or without Ppargc1a-siRNA, and J VD( + ) HSC-recipient macrophages with or without Jarid2-siRNA (n = 6/group). K–M Fetal HSCs from WT or miR-106b−/− animals under VD(−) or VD(+) conditions were transplanted into VD( + ) WT recipients. K Glucose tolerance tests and L insulin tolerance tests (n = 8/group). M Insulin-stimulated 2-DG uptake in 3T3-L1 adipocytes co-cultured with peritoneal macrophages from WT or miR-106b−/− animals transplanted with VD(−) or VD(+) HSCs (n = 6/group). Data presented as mean ± SEM. A, C, D, F, H, I, J *P < 0.05; ***P < 0.001 by two-tailed unpaired t test. G, M *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA followed by Tukey’s multiple comparison test. K, L *P < 0.05; **P < 0.01; ***P < 0.001 VD(−) WT vs. all and †P < 0.05; †††P < 0.001 for VD( + ) WT vs. VD(−) miR-106b−/−. Actual P values shown in source data file.

false