rabbit anti sk1  (Affinity Biosciences)

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ca 2+ /Calmodulin-Dependent Protein Kinase II (CaMKII) Increases Small-Conductance Ca 2+ -Activated K + Current in Patients with Chronic Atrial Fibrillation

doi: 10.12659/MSM.909684

Figure Lengend Snippet: qRT-PCR and Western blotting results showing KCNN 1–3 mRNA levels and K Ca 2.1/2.2/2.3 proteins levels in the atria of SR controls (n=20) and AF patients (n=32). ( A ) mRNA levels of KCNN1, KCNN2, and KCNN3 in SR and AF. ( B ) mRNA expression differences of KCNN1, KCNN2, and KCNN3 in SR group. ( C ) mRNA expression differences of KCNN1, KCNN2, and KCNN3 in AF group. ( D ) K Ca 2.1–2.3 (SK1–3) proteins expression changes in SR (n=20) and AF (n=32). * P <0.05 vs. SR.

Article Snippet: The PVDF membrane was incubated with rabbit polyclonal anti-KCa 2.1 (SK1), anti-KCa 2.2 (SK2), anti-KCa 2.3 (SK3, Alomone Labs, Jerusalem, Israel) (dilution 1: 500), rabbit polyclonal anti-CaM (Santa Cruz Biotechnology, Santa Cruz, USA) (dilution 1: 1000), rabbit polyclonal anti-CaMKII (Abcam, Cambridge, UK) (dilution 1: 1000), rabbit polyclonal anti-pCaMKII (Thr 286 , Cell signaling, USA) (dilution 1: 1000), anti-pCaMKII (Thr 287 , Abcam, Cambridge, UK) (dilution 1: 1000), and rabbit polyclonal anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, USA) (dilution 1: 2000) overnight at 4ºC.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: The FASEB Journal

Article Title: Targeting sphingosine kinase 1 in p53KO thymic lymphoma

doi: 10.1096/fj.202301417r

Figure Lengend Snippet: FIGURE 4 SK1 inhibition decreases cell survival in mouse p53KO thymic lymphoma cells. (A) Mouse p53KO thymic lymphoma cells were treated with vehicle (black line) or PF543 0.5 μM (red line) and cells were counted by trypan blue exclusion every day for 4 days. Data shown are mean ± SD (n = 4). Statistical analysis: 2-way ANOVA **p < .001; ***p < .0005; ****p < .0001. (B) Cell viability was measured by CCK-8 assay at day 2 after treatment with vehicle (control, black bar) or PF543 0.5 μM (red bar). Data shown are mean ± SD (n = 4). Statistical analysis: Unpaired t test with Welch's correction **p < .001. (C) Sphingosine kinase activity was measured after 24 h of treatment with vehicle (control, black bar) or PF543 0.5 μM (red bar). C17-Sphingosine was added to the cells 20 min before cell collection, and after lipid extraction C17-S1P was measured by LC/MS. Data shown are mean ± SD (n = 2). Statistical analysis: Unpaired t test with Welch's correction *p < .005. (D) Mouse p53KO thymic lymphoma cells treated with vehicle (black line) or PF543 0.5 μM (red line) were processed for sphingolipid analysis by LC MS/MS. Values shown are the mean ± SD (n = 2). Statistical analysis for Sph and S1P: 2-way ANOVA, Sidak's multiple comparison test, *p < .05. Statistical analysis: Unpaired t test with Welch's correction *p < .005. Statistical analysis for total ceramide: Unpaired t test with Welch's correction *p < .005.

Article Snippet: Primary antibodies used for Western blot included SK1 (1:1000, Cat #12071, Cell Signaling Technology), SK2 (1:1000, ab37977, Abcam), Parp1 (1:2000, Cat#9542, Cell Signaling Technology), cleaved caspase 3 (1:1000, Cat#9661, Cell Signaling Technology), caspase 8 (1:1000, Cat#4790, Cell Signaling Technology), caspase 7 (1:1000, Cat#12827, Cell Signaling Technology), caspase 9 (1:1000, Cat#95020, Cell Signaling Technology), p16 (1:1000, ab211542, Abcam), p21 (1:1000, sc- 6246, Santa Cruz Biotechnology), Beta- Actin (1:25000, Cat. #A5441, Sigma Aldrich).

Techniques: Inhibition, CCK-8 Assay, Control, Activity Assay, Extraction, Liquid Chromatography with Mass Spectroscopy, Comparison

Journal: The FASEB Journal

Article Title: Targeting sphingosine kinase 1 in p53KO thymic lymphoma

doi: 10.1096/fj.202301417r

Figure Lengend Snippet: FIGURE 5 Pharmacological inhibition of SK1 controls thymic lymphoma growth in Trp53KO mice. (A) Blood samples from mice treated with vehicle (black bar) or PF543 (red bar, 10 mg/kg of body weight) after 2 h of IP injection were processed for sphingolipid analysis by LC MS/MS. Values shown are the mean ± SD (n = 2). Statistical analysis: 2-way ANOVA, Sidak's multiple comparison test, *p < .005; **p < .001. (B) Body weight was assessed every two days in mice treated with vehicle (black line) or PF543 (red line, 10 mg/kg of body weight) with 2 IP injections per day for 20 days. (C) Concentration of liver enzymes was evaluated in blood samples from mice treated with Vehicle (black bar) or PF543 (red bar, 10 mg/kg of body weight) with 2 IP injections per day for 20 days. (D) Blood Urea Nitrogen (BUN) was measured in mice treated with Vehicle (black bar) or PF543 (red bar, 10 mg/kg of body weight) with 2 IP injections per day for 20 days. (E) Blood counts were performed on whole blood from mice treated with vehicle (black circle, n = 5) or PF543 (red circle, 10 mg/kg of body weight, n = 5) with 2 IP injections per day for 20 days. Blood was analyzed for lymphocytes and white blood cells (WBCs) number. (F) Protocol of treatment with PF543 in Trp53KO mice once thymic lymphoma was detected by ultrasound imaging. (G) Tumor size of thymic lymphoma from Trp53KO mice treated with vehicle (control, black, n = 6) or PF543 (red, n = 6) accordance to protocol shown in Figure 5E. Statistical analysis: 2-way ANOVA, Sidak's multiple comparison test, *p < .005; ***p < .0005.

Article Snippet: Primary antibodies used for Western blot included SK1 (1:1000, Cat #12071, Cell Signaling Technology), SK2 (1:1000, ab37977, Abcam), Parp1 (1:2000, Cat#9542, Cell Signaling Technology), cleaved caspase 3 (1:1000, Cat#9661, Cell Signaling Technology), caspase 8 (1:1000, Cat#4790, Cell Signaling Technology), caspase 7 (1:1000, Cat#12827, Cell Signaling Technology), caspase 9 (1:1000, Cat#95020, Cell Signaling Technology), p16 (1:1000, ab211542, Abcam), p21 (1:1000, sc- 6246, Santa Cruz Biotechnology), Beta- Actin (1:25000, Cat. #A5441, Sigma Aldrich).

Techniques: Inhibition, Injection, Liquid Chromatography with Mass Spectroscopy, Comparison, Concentration Assay, Imaging, Control

Journal: The FASEB Journal

Article Title: Targeting sphingosine kinase 1 in p53KO thymic lymphoma

doi: 10.1096/fj.202301417r

Figure Lengend Snippet: FIGURE 7 Targeting SK1 in Trp53KO mice controls thymic lymphoma growth by increased apoptosis. (A) Western blot analysis for cleaved caspase 3 and Parp1 protein expression in lymphoma tissue samples from Trp53KO Sphk1+/+ Mx1Cre+ (control) and Trp53KO Sphk1fl/fl Mx1Cre+ mice at day 20 after initiation of treatment. Actin served as internal loading control. (B) Immunohistochemistry staining for cleaved caspase 3 of tumor tissue sections dissected from Trp53KO mice treated with vehicle or PF543 at day 10 after tumor treatment initiation. Representative images from three mice per each condition are shown. Quantification of the number of cleaved caspase 3 positive cells per mm2 is shown on the right. Statistical analysis: Unpaired t test with Welch's correction *p < .005.

Article Snippet: Primary antibodies used for Western blot included SK1 (1:1000, Cat #12071, Cell Signaling Technology), SK2 (1:1000, ab37977, Abcam), Parp1 (1:2000, Cat#9542, Cell Signaling Technology), cleaved caspase 3 (1:1000, Cat#9661, Cell Signaling Technology), caspase 8 (1:1000, Cat#4790, Cell Signaling Technology), caspase 7 (1:1000, Cat#12827, Cell Signaling Technology), caspase 9 (1:1000, Cat#95020, Cell Signaling Technology), p16 (1:1000, ab211542, Abcam), p21 (1:1000, sc- 6246, Santa Cruz Biotechnology), Beta- Actin (1:25000, Cat. #A5441, Sigma Aldrich).

Techniques: Western Blot, Expressing, Control, Immunohistochemistry, Staining

Journal: Journal of Lipid Research

Article Title: 1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase

doi: 10.1016/j.jlr.2021.100154

Figure Lengend Snippet: dSA regulates SK1 levels. A: Effect of inhibiting de novo sphingolipid synthesis on SK1 levels following serine starvation. Myriocin (100 nM) was added 6 h before the medium change, and cells were incubated for 24 h before processing and analysis of SK1 by Western blot. B: Effect of the proteasomal inhibitor bortezomib (BTZ) on SG starvation-induced SK1 proteolysis. C: SK1 protein levels following treatment with Sph and dSA. Both caspase (ZVAD-fmk) and proteosomal (BTZ) inhibitors were added concurrently with either Sph or dSA and incubated for 24 h before SK1 was analyzed using Western blot. D: Effect of SG starvation on SK1 ubiquitination. Cells were transfected with 1 μg of His-Ub containing plasmid for 24 h before the media was changed. Cells were incubated for a further 24 h before lysis and purification of His-Ub and detection of SK1 by Western blot. For comparison, treatment with SK1-inhibitors PF543 and SKi-II that cause SK1 ubiquitination is also shown. E: Protein lipid overlay comparing SK1 protein binding to Sph, dSA, or phosphatidylcholine (PC). Sph (positive control), dSA, and PC (negative control) were spotted onto nitrocellulose before blocking and overlay of SK1 protein. SK1 was identified as described for Western blotting.

Article Snippet: Equal amounts of proteins were loaded and separated on precast Novex 4–20% SDS–PAGE midi gels (Life Technologies, Carlsbad, CA), transferred to nitrocellulose membranes, and probed with antibodies against SK1 (12071) and PHGDH (13428S) from Cell Signaling Technology (Danvers, MA); and β-actin (A5441) from Sigma.

Techniques: Incubation, Western Blot, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Lysis, Purification, Comparison, Protein Binding, Positive Control, Negative Control, Blocking Assay

Journal: Journal of Lipid Research

Article Title: 1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase

doi: 10.1016/j.jlr.2021.100154

Figure Lengend Snippet: Serine-deficient diet alters the levels of SK1 and sphingolipids in tumor xenografts implanted in mice. Mice were implanted subcutaneously with HCT116 cells (5 × 10 6 ) in the left hind flank and fed either control or SG-deficient diet until the control tumors had reached their maximum allowable volume. Tumors were analyzed for (A) growth, (B) SK1 levels, (C) levels of dSA, (D) levels of dihydrosphingosine (dhSph) and Sph, and (E) levels of dihydroS1P(dhS1P) and S1P via LC/MS and normalized to total lipid phosphate. Results represent mean ± SEM (n = 10). ∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ P < 0.0005.

Article Snippet: Equal amounts of proteins were loaded and separated on precast Novex 4–20% SDS–PAGE midi gels (Life Technologies, Carlsbad, CA), transferred to nitrocellulose membranes, and probed with antibodies against SK1 (12071) and PHGDH (13428S) from Cell Signaling Technology (Danvers, MA); and β-actin (A5441) from Sigma.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Lipid Research

Article Title: 1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase

doi: 10.1016/j.jlr.2021.100154

Figure Lengend Snippet: Scheme representing the biological effects of serine starvation on sphingolipid synthesis and their consequences. Serine deprivation leads to substitution of alanine by SPT to generate 1-dSA. Binding of dSA to SK1 leads to KLHL5 mediated proteolysis of SK1, allowing for Sph accumulation. Increased Sph leads to increased ROS, which increases PHGDH expression while decreasing PKM2 activity. These lead to increased serine synthesis, allowing for increased cell growth. Serine palmitoyl transferase (SPT), deoxysphinganine (dSA), sphingosine (Sph), Sph kinase 1 (SK1), Sph 1-phosphate (S1P), oxygen consumption rate (OCR), reactive oxygen species (ROS), phosphoglycerate dehydrogenase (PHGDH), pyruvate kinase m2 (PKM2). Dotted line represents speculated consequence.

Article Snippet: Equal amounts of proteins were loaded and separated on precast Novex 4–20% SDS–PAGE midi gels (Life Technologies, Carlsbad, CA), transferred to nitrocellulose membranes, and probed with antibodies against SK1 (12071) and PHGDH (13428S) from Cell Signaling Technology (Danvers, MA); and β-actin (A5441) from Sigma.

Techniques: Binding Assay, Expressing, Activity Assay