ISL, EZ (purity > 95%), and (R)-Mevalonic acid lithium salt were purchased from Sigma, New York, NY, USA. Niemann-Pick C1-like 1, β-actin, β-tubulin (antibodies were purchased from ABclonal, Wuhan, China), fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM) and cell lysis buffer (10×), secondary antibody horseradish peroxidase-conjugated anti-rabbit (L3012), ultra-enhanced chemiluminescence detection reagent, Filipin bacteriocin, and BCA protein average/concentration detection kit were all purchased from Meilun Biotechnology (Dalian, China). Sodium taurocholate hydrate and lovastatin were purchased from Maclean’s, NBD-labeled cholesterol (from J&K scientific, Beijing, China).
β tubulin
Manufactured by ABclonal
Sourced in China, United States
β-tubulin is a protein that forms part of the cytoskeleton in eukaryotic cells. It is a component of microtubules, which are essential for various cellular processes such as cell division, intracellular transport, and cell motility.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Gapdh, by ABclonal (3 mentions)
β actin, by ABclonal (2 mentions)
Gapdh, by Proteintech (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
R mevalonic acid lithium salt, by Merck Group (1 mentions)
Bca protein average concentration detection kit, by Meilun (1 mentions)
Fbln5, by ABclonal (1 mentions)
Bca protein analysis kit, by Thermo Fisher Scientific (1 mentions)
Mmp13, by Abcam (1 mentions)
Hrp labeled secondary antibody, by ZSGB-BIO (1 mentions)
Total erk1 2, by ABclonal (1 mentions)
Phospho erk1 2, by ABclonal (1 mentions)
Col 2, by Thermo Fisher Scientific (1 mentions)
26 protocols using β tubulin
1
Cholesterol Uptake and Metabolism
2
Protein Expression Analysis of Cell Lines
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Collected cells (GES, AGS, BGC-823, HGC-27, and MKN-28) were lysed on ice with phosphatase and protein inhibitors in RIPA buffer for 30 min, then centrifuged at 13,000 rpm for 15 min. The cell debris was removed, the supernatant was obtained using a pipette, a certain amount of 5× loading buffer was added, and samples were heated at 100 °C for 10 min. The BCA protein analysis kit (Thermo Scientific, Waltham, MA, USA) was used to quantitatively detect the protein concentration. The protein was dissolved using 12% SDS-PAGE, the protein was transferred to a PVDF membrane at low temperatures, 5% skimmed milk powder was used for blocking for 2 h, and the membrane was incubated with the primary antibody overnight at 4 °C (FBLN5: 1:1000, ABclonal, USA; β-Tubulin: 1:1000, ABclonal, USA). The membrane was incubated for 1 h the next day with a horseradish peroxidase-labeled secondary antibody (1:5000). The ECL kit (Thermo Scientific, USA) was used to assess the expression of individual proteins. The experiment was conducted in triplicate (Figure S1 of Supplementary Materials ).
3
Protein Extraction and Western Blot Analysis
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Minced tissues were subjected to extract protein using RIPA solution supplemented with a protease inhibitor cocktail and protein phosphatase inhibitor cocktail. The protein concentration was quantified using a BCA protein quantitation kit (Thermo Fisher Scientific, USA). The 30 μg of proteins were separated via 10~12% SDS-PAGE and then transferred to nitrocellulose (NC) membranes. After blocking, the membranes were incubated with the following primary antibodies against GnRH (1:1000, Abcam, UK), Kisspeptin-1 (1:800, Proteintech, USA), GPR54 (1:2500, Poteintech), total-Sp1 (1:900, ABclonal, China), phospho-Sp1 (1:1200, ABclonal), total-ERK1/2 (1:1500, ABclonal), phospho-ERK1/2 (1:1500, ABclonal), MMP-13 (1:1200, Abcam), Col-X (1:1500, ABclonal), Col-Ⅱ (1:1200, Thermo Fisher Scientific) and β-tubulin (1:4000, ABclonal) overnight at 4 ˚C. Finally, the membranes were incubated with HRP-labeled secondary antibodies (1:5000, ZSGB-BIO, China). The protein band was visualized and measured with an ECL kit (Merck Millipore, USA). ImageJ software was applied for the analysis of band intensity.
4
Protein Extraction and Western Blot Analysis
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NSCLC cells were lysed in lysis buffer (RIPA buffer and 1% PMSF) (Beyotime) on
ice. Then, the cell lysate was sonicated for 15 s and centrifuged at 12 000
r/min for 15 min. The protein concentrations were quantified using the BCA
reagent (Applygen Technologies, Beijing, China). The whole-cell extracts were
boiled for 5 min in sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) buffer (Beyotime). The samples were separated in 8–12%
SDS–polyacrylamide gels and electrotransferred to polyvinylidene difluoride
membranes. After blocking with 8% non-fat milk in Tris-buffered saline Tween,
the membranes were probed overnight with the following antibodies at 4°C: GAPDH
(Abcam), β-tubulin (ABclonal, Seoul, Korea), β-actin (Abcam), and KRT8
(ABclonal). The membranes were then incubated with horseradish
peroxidase-conjugated goat anti-rabbit and goat anti-mouse (Abcam) secondary
antibodies at RT for 1 h. Finally, an enhanced chemiluminescence detection kit
(Beyotime) was used to visualize the immunoreactive proteins.7 (link)
ice. Then, the cell lysate was sonicated for 15 s and centrifuged at 12 000
r/min for 15 min. The protein concentrations were quantified using the BCA
reagent (Applygen Technologies, Beijing, China). The whole-cell extracts were
boiled for 5 min in sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) buffer (Beyotime). The samples were separated in 8–12%
SDS–polyacrylamide gels and electrotransferred to polyvinylidene difluoride
membranes. After blocking with 8% non-fat milk in Tris-buffered saline Tween,
the membranes were probed overnight with the following antibodies at 4°C: GAPDH
(Abcam), β-tubulin (ABclonal, Seoul, Korea), β-actin (Abcam), and KRT8
(ABclonal). The membranes were then incubated with horseradish
peroxidase-conjugated goat anti-rabbit and goat anti-mouse (Abcam) secondary
antibodies at RT for 1 h. Finally, an enhanced chemiluminescence detection kit
(Beyotime) was used to visualize the immunoreactive proteins.7 (link)
5
Evaluation of Gefitinib and Sunitinib on HUVEC and Lung Cancer Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from Sciencell (CA, USA), and human lung cancer cell lines, HCC827 and H3255, were purchased from American Type Culture Collection (VA, USA). HUVECs were cultured in endothelial cell medium (ECM) kit (Sciencell) containing 5% fetal bovine serum (FBS) and 1% endothelial cell growth supplement. The human lung cancer cell lines were cultured in medium 1640 (Thermo Fisher Scientific, MA, USA) containing 10% FBS (Thermo Fisher Scientific), and 100 U/mL penicillin and 0.1 mg/mL streptomycin were added to the medium. All cells were cultured in an incubator at 37°C with 5% CO2.
Gefitinib and sunitinib were purchased from Selleck (TX, USA). CM082 was provided by Betta Pharmaceutica Co., Ltd. (Hangzhou, China). Recombinant human VEGF was purchased from R&D Systems (MN, USA). Antibodies for VEGFR2, p‐VEGFR2, ERK, p‐ERK, AKT, p‐AKT, p‐STAT3, and STAT3 were purchased from Cell Signaling Technology (MA, USA). Antibodies for VEGF‐A, CD31, Ki‐67, β‐actin, and β‐tubulin, and horseradish peroxidase‐conjugated antirabbit/mouse IgG antibodies, were purchased from ABclonal Technology (Wuhan, China).
Gefitinib and sunitinib were purchased from Selleck (TX, USA). CM082 was provided by Betta Pharmaceutica Co., Ltd. (Hangzhou, China). Recombinant human VEGF was purchased from R&D Systems (MN, USA). Antibodies for VEGFR2, p‐VEGFR2, ERK, p‐ERK, AKT, p‐AKT, p‐STAT3, and STAT3 were purchased from Cell Signaling Technology (MA, USA). Antibodies for VEGF‐A, CD31, Ki‐67, β‐actin, and β‐tubulin, and horseradish peroxidase‐conjugated antirabbit/mouse IgG antibodies, were purchased from ABclonal Technology (Wuhan, China).
6
Quantification of Myogenic Protein Expression
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To confirm the expression profiles of target genes, we quantified their protein levels using a classical WB assay. Firstly, the total proteins from in vitro cultured cells were extracted using a radioimmunoprecipitation assay kit (RIPA) (Beyotime, Shanghai, China) and quantified by the bicinchoninic acid assay kit (BCA) (Beyotime, China). Secondly, the qualified protein samples (~20 μg per sample) were loaded separately in polyacrylamide gel electrophoresis and transferred to Polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Thirdly, PVDF membranes containing total proteins were incubated with primary anti-rabbit myogenic differentiation 1 (MyoD) (1:1000) and myosin heavy chain (MyHC) (1:1000) (Abclonal, Wuhan, China) at 4 °C overnight, and secondary antibody IgG (1:2000) (Abclonal, China) for 2 h. Finally, after adding Horse Radish Peroxidase (HRP) (Bio-Rad, Hercules, CA, USA), the protein bands were detected and analyzed using electrochemiluminescence (ECL) (Pierce, Appleton, WI, USA) and Image Software (version 6.0.1) with β-Tubulin (1:1000) (Abclonal, China) as a loading control.
7
Protein Extraction and Western Blot Analysis
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After desired treatments as specified, cells were washed twice with PBS and lysed in buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycer-ophosphate, 1 mM sodium vanadate, 1 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) on ice. Equal amounts of protein (30 μg) were loaded onto 8% SDS-PAGE gels. Western detection was performed using a chemiluminescence Western blot scanner (ChampGel 7000, SAGECREATION). For protein detection, the following antibodies were used: ACC1 (#21923-1-AP; Proteintech), ACC2 (#8578; Cell Signaling), phosphorylation ACC (#3661; Cell Signaling), CD36 (#18836-1-AP; Proteintech), LAMP2 (#66301-1-Ig; Proteintech), TOM20 (ab56783; Abcam), GAPDH (#60004-1-Ig; Proteintech), Lamin B1 12987-1-AP(proteintech), RAC1 (#24072-1-AP; Proteintech), β-Actin (#AC026; Abclonal), β-tubulin (#AC008; Abclonal), goat anti-rabbit IgG/HRP (#115-035-003; Jackson) and goat anti-mouse IgG/HRP (#111-035-003; Jackson).
8
Western Blot Analysis of Autophagy and Apoptosis
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Cells were lysed using RIPA lysis buffer (Beyotime, China). Protein samples were electrophoresed in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with TBST containing 5% nonfat milk, membranes were incubated with primary antibodies against LC3 (Abcam, UK), p62 (Abcam, UK), caspase-3 (Cell signaling technology, USA), GAPDH (Cell signaling technology, USA), bcl-2 (ABclonal, China) and β-tubulin (ABclonal, China) at 4°C overnight. Then, the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Beyotime, China) for 1 h, and detected by Tanon Image Lab Software.
9
Immunoprecipitation and Western Blot Analysis
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Cells were treated with cell lysis buffer for Western and IP (Beyotime). The indicated antibodies were added to cell lysates or supernatant, followed by incubation with gentle rocking overnight at 4°C. Then, protein G agarose beads were added to cell lysates, which was incubated with gentle rocking for 4 h at 4°C. Next, samples were eluted in SDS‐PAGE sample loading buffer and boiled. For immunoprecipitation, anti‐FLAG (AE005; ABclonal), STING (19851‐1‐AP; Proteintech) and MLKL (PA5‐102810; Invitrogen) antibodies were used.
The primary antibodies were used including STING (19851‐1‐AP; Proteintech), hp‐STING (S366, 50907; CST), mp‐STING (S365, 72971; CST), p‐TBK1 (S172; CST), TBK1 (38066; CST), IRF3 (4302; Cell Signaling), p‐IRF3 (S396, 29047; CST), mp‐RIPK3 (T231+S232, ab222320; Abcam), mp‐MLKL(S345, ab196436; Abcam), hp‐RIPK3 (S227, ab209384; Abcam), hp‐MLKL (S358, ab187091; Abcam), hMLKL (A19685; ABclonal), mMLKL (37705; CST), hRIPK3 (86671; CST), mRIPK3 (15828; CST), mp‐RIPK1 (S166, 31122; CST), RIPK1 (3493; CST), LC3 (12741; CST), P62 (A7758; ABclonal), β‐actin (4970; CST), β‐Tubulin (AC021; ABclonal), GAPDH (60004‐1‐Ig; Proteintech), FLAG‐tag (AE063; ABclonal), HA‐tag (ab236632; Abcam), MYC‐tag (AE070; ABclonal) and GFP‐tag (AE012; ABclonal).
The primary antibodies were used including STING (19851‐1‐AP; Proteintech), hp‐STING (S366, 50907; CST), mp‐STING (S365, 72971; CST), p‐TBK1 (S172; CST), TBK1 (38066; CST), IRF3 (4302; Cell Signaling), p‐IRF3 (S396, 29047; CST), mp‐RIPK3 (T231+S232, ab222320; Abcam), mp‐MLKL(S345, ab196436; Abcam), hp‐RIPK3 (S227, ab209384; Abcam), hp‐MLKL (S358, ab187091; Abcam), hMLKL (A19685; ABclonal), mMLKL (37705; CST), hRIPK3 (86671; CST), mRIPK3 (15828; CST), mp‐RIPK1 (S166, 31122; CST), RIPK1 (3493; CST), LC3 (12741; CST), P62 (A7758; ABclonal), β‐actin (4970; CST), β‐Tubulin (AC021; ABclonal), GAPDH (60004‐1‐Ig; Proteintech), FLAG‐tag (AE063; ABclonal), HA‐tag (ab236632; Abcam), MYC‐tag (AE070; ABclonal) and GFP‐tag (AE012; ABclonal).
10
Investigating Protein Interactions
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Co-immunoprecipitation (Co-IP) and immunoprecipitation (IP) were conducted using the IP/Co-IP kit (ABsin) according to the manufacturer's instructions. The primary anti-bodies used in this assay included β-catenin (ABclonal), YWHAZ (Proteintech), Ubiquitin (CST), β-tubulin (ABclonal).
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