NP cells were treated with 10 ng/mL leptin (Sigma-Aldrich, Oakville, ON, Canada) for 5 min, 10 min, 30 min and 24 h, respectively. Untreated cells were used as controls. Cell lysates were subject to SDS-PAGE and transferred to PVDF membranes (Millipore, Boston, MA, USA). Primary antibodies against the following proteins were used: LIMK1, p-LIMK1, cofilin-2, p-cofilin-2 (Abcam, Cambridge, UK) and GAPDH (Abcam, Cambridge, UK). HRP-conjugated secondary antibodies were used. Signal was detected using ECL kit (Millipore, Boston, MA, USA).
Limk1
Manufactured by Abcam
Sourced in United Kingdom
LIMK1 is a protein-coding gene that produces the LIM domain kinase 1 enzyme. This enzyme is involved in the regulation of actin cytoskeleton dynamics, which play a crucial role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Leptin, by Merck Group (1 mentions)
P limk1, by Abcam (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Prdm14, by Abcam (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
P pak1, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Caspase 3, by Abcam (1 mentions)
Immunohistochemistry kit, by Maixin Group (1 mentions)
Destrin, by Abcam (1 mentions)
Vimentin, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
3 protocols using limk1
1
Leptin-Induced LIMK1 and Cofilin Signaling
2
Western Blot Protein Extraction and Analysis
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Proteins from whole-cell lysates for western blotting were extracted with mammalian protein extraction reagent (cat. no. 78501; Thermo Scientific, Rockford, IL, USA), resolved on SDS–PAGE and transferred to polyvinylidene fluoride (PVDF; cat. no. IPVH00010; EMD Millipore) membrane. The membranes were then blocked with 5% bovine serum albumin (BSA; cat. no. A7906; Sigma-Aldrich, St Louis, MO, USA) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated with primary antibody for 1 h at room temperature, followed by rinsing of unbound antibody with TBST and incubating the membranes with appropriate horseradish peroxidase-conjugated secondary antibodies. Primary antibodies for Cdc42 (cat. no. ab64533; Abcam, Cambridge, UK), E-cadherin (cat. no. 3195S, Cell Signaling, Danvers, MA, USA), vimentin (cat. no. 5741S; Cell Signaling), p-PAK1 (p-21-activated kinase 1) (cat. no. ab40852; Abcam), LIMK1 (cat. no. ab95186; Abcam), prdm14 (cat. no. ab91587; Abcam), caspase-3 (cat. no. C8487; Sigma-Aldrich) and β-actin (cat. no. A1978; Sigma-Aldrich) were used. Protein expression was detected using SuperSignal West Femto Maximum Sensitivity Substrate (cat. no. 34095; Thermo Scientific) and the membranes were imaged for chemiluminescence using the ImageQuant LAS 4000 Imager (GE Healthcare Life Sciences, Bio-Sciences, Pittsburgh, PA, USA).
3
Immunohistochemical Analysis of Cytoskeletal Proteins
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The paraffin sections were deparaffinized, followed by hydration, using an immunohistochemistry kit (Maixin Biotech. Co., Fuzhou, China) according to the manufacturer instructions. LIMK1, destrin, E-cadherin, vimentin, Ki-67, and CD34 antibodies (Abcam, Cambridge, UK) were incubated overnight at 4 °C, and normal rabbit immunoglobulin G was considered as a negative control. The specimens were visualized with 3,3′-diaminobenzidine and counterstained with hematoxylin. The staining was brown or tan and located in the cytoplasm or nucleus. The scores according to the degree of positive staining and the percentage of stained cells were as follows: 0, no coloring; 1, light brown; 2, dark brown; 0, staining cells < 5%; 1, staining cells between 5% and 25%; 2, staining cells between 26% and 50%; 3, staining cells > 50%. The score was obtained by adding the intensity and reactivity. The scores < 2 represented low expression, the scores ≥ 2 represented the high expression.
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