2D gel electrophoresis was performed as described previously24 (link). Proteins (30–50 μg/sample) were separated by isoelectric focusing using strips that range from pH 3 to 10, then loaded on 10% SDS-PAGE gels, and transferred to a PVDF membrane. Membranes were blocked with 5% dry fat-free milk solution in PBST (PBS containing 0.1% Tween 20) and incubated overnight at 4 °C with the indicated antibodies: Rbfox2 (abcam #57154; 1:500), GAPDH (abcam #9484; 1:2500). Membranes were washed with PBST 3 × 15 minutes and incubated with HRP-labeled secondary antibody for 2 hrs at room temperature. HRP activity was determined using Immobilon Western Chemiluminescent reagent (Millipore P90720) or SuperSignal West Femto Chemiluminescent (Pierce PI34095) HRP substrate followed by exposure to x-ray film.
Immobilon western chemiluminescent reagent
Manufactured by Merck Group
Sourced in United States
Immobilon Western chemiluminescent reagents are a set of laboratory equipment used for the detection and quantification of proteins in Western blot analysis. These reagents enable the visualization of target proteins on a membrane by producing a luminescent signal upon interaction with the proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ibright imaging system, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Sc 8019, by Santa Cruz Biotechnology (2 mentions)
Gtx118000, by GeneTex (2 mentions)
Tris glycine acrylamide gel, by Thermo Fisher Scientific (2 mentions)
Hatf00010, by Merck Group (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Hrp linked goat anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Bs 2188r, by Bioss Antibodies (1 mentions)
Gapdh, by Bioss Antibodies (1 mentions)
Vimentin, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Paris kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Proteintech (1 mentions)
Ripa lysis buffer, by Selleck Chemicals (1 mentions)
12 protocols using immobilon western chemiluminescent reagent
1
Proteomic Analysis by 2D Gel Electrophoresis
2
Immunoblot Analysis of B Cell Proteins
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B220+ B cells were sorted from the spleen of AID+ ki/+ and WT mice, or Mb1+ ki/+ and WT mice. Then the RIPA extracts were fractionated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels, electroblotted to polyvinylidene difluoride (PVDF) membranes and reacted with anti-c-MYC (Cell Signaling Technology, 9402), anti-BCL2 (Cell Signaling Technology, 3498) and anti-β-actin (Cell Signaling Technology, 4967) antibodies, and followed by HRP linked goat anti-rabbit antibody (Cell Signaling, 7074). HRP activity was determined using Immobilon Western Chemiluminescent reagent (Millipore, P90720).
3
Jak1/STAT3 Signaling Pathway Analysis
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MK45 cells were seeded in six-well tissue culture plate (cell density, 400 × 103 per well) in 100 µl of RPMI 1640 medium supplemented with 10% FBS, 1% l -glutamine, and 1% penicillin and streptomycin at 37°C and 5% CO2. Cells were serum-starved for 24 h and then incubated with LIF (10 ng/ml) and EC359 (25 and 100 nM), alone or in combination, for 48 h. Total lysates were prepared by homogenization of MKN45 cells in Ripa buffer containing phosphatase and protease inhibitors. Protein extracts were electrophoresed on 12% acrylamide Tris-Glycine gel (Invitrogen), blotted to nitrocellulose membrane, and then incubated overnight with primary Abs against Jak1 (1:500; sc-7228, Santa Cruz Biotechnology), phospho-Jak1 (1:1,000; GTX25493, GeneTex), STAT3 (1:500; sc-8019, Santa Cruz Biotechnology), phosho-Stat3 (1:1,000; GTX118000, GeneTex), and Gapdh (1:1,000; bs2188R, Bioss Antibodies). Primary Abs were detected with the HRP (horseradish peroxidase)-labeled secondary Abs. Proteins were visualized by Immobilon Western Chemiluminescent Reagent (MilliporeSigma) and iBright Imaging Systems (Invitrogen). Quantitative densitometry analysis was performed using ImageJ software. The degree of JAK1 and STAT3 phosphorylation was calculated as the ratio between the densitometry readings of p-Jak1/Jak1 and p-STAT3/STAT3, respectively.
4
Mifepristone Modulates STAT3 Phosphorylation
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MIA-PaCa 2 cells were seeded in a 6-well tissue culture plate (cell density 1.5 × 106/well) in DMEM medium supplemented with 10% foetal bovine serum, 1% L-glutamine, and 1% penicillin and streptomycin at 37 °C and 5% CO2. Cells were serum-starved for 24 h and then incubated with LIF (10 ng/mL) alone or plus mifepristone (10, 20, 50 µM) for 10 min. Total lysates were prepared by homogenization of MIA-PaCa2 cells in Ripa buffer containing phosphatase and protease inhibitors. Protein extracts were electrophoresed on 12% acrylamide Tris-Glycine gel (Invitrogen), blotted to the nitrocellulose membrane, and then incubated overnight with primary Abs against STAT3 (sc-8019 1:500; Santa Cruz Biotechnology) and phosho-Stat3 (GTX118000 1:1000; Genetex). Primary Abs were detected with the HRP-labelled secondary Abs. Proteins were visualized by Immobilon Western Chemiluminescent Reagent (MilliporeSigma) and iBright Imaging Systems (Invitrogen). Quantitative densitometry analysis was performed using ImageJ software. The degree of STAT3 phosphorylation was calculated as the ratio between the densitometry readings of p-STAT3/STAT3.
5
STAT3 Activation in MIA-PaCa2 Cells
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MIA-PaCa 2 cells were seeded in 6-well tissue culture plate (cell density 1.5 * 106/well) in DMEM complete medium. After serum starving, cells were incubated with LIF (10 ng/mL) alone or plus BAR502 (10 µM) for 10 min. Total lysates were prepared by homogenization of MIA-PaCa2 cells in RIPA buffer containing phosphatase and protease inhibitors. Protein extracts were electrophoresed on 12% acrylamide Tris-Glycine gel (Invitrogen), blotted to nitrocellulose membrane, and then incubated overnight with primary Abs against GAPDH (bs2188R 1:1000; Bioss antibodies), STAT3 (sc-8019 1:500; Santa Cruz Biotechnology), Vimentin (ab92547 1:1000;Abcam), phosho-Stat3 (GTX118000 1:1000; Genetex). Primary Abs were detected with the HRP-labeled secondary Abs. Proteins were visualized by Immobilon Western Chemiluminescent Reagent (MilliporeSigma) and iBright Imaging Systems (Invitrogen). Quantitative densitometry analysis was performed using ImageJ software. The degree of STAT3 phosphorylation was calculated as the ratio between the densitometry readings of Vimentin/GAPDH and p-STAT3/STAT3.
6
Protein Fractionation and Western Blot Analysis
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Whole cell protein was isolated using Pierce IP Lysis Buffer (Thermo Fisher Scientific, United States). The PARIS Kit (AM1921, Invitrogen) was used to separate nuclear and cytoplasmic protein fractions in GBM cells. Protein concentrations were quantified using the Micro BCA Protein Assay Kit (23229, Thermo Scientific). Purified proteins were boiled with 4 × loading buffer, and denatured protein samples were separated by SDS-PAGE on 10% polyacrylamide gels. Then, samples were transferred to NC membranes (HATF00010, Millipore). After blocking with 5% non-fat milk for 1 h, membranes were probed with appropriate primary antibodies overnight at 4°C. The next day, membranes were washed with PBS/Tween (PBST) and incubated with appropriate secondary antibodies for approximately 1 h at room temperature. Protein bands were visualized by Immobilon Western chemiluminescent reagents (WBKLS0500, Millipore). Information for utilized primary antibodies is shown in Supplementary Table S3 .
7
Western Blot Analysis of CRABP2
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The specific method of western blot was consistent with our previous research [31 (link)]. Cell lysates were prepared using RIPA lysis buffer (Bimake, Houston, Texas, USA), and the proteins in cell lysates were separated using 10% SDS–PAGE and transferred into PVDF membranes (Millipore, Billerica, MA, USA). After being blocked with skimmed dry milk, PVDF membranes were incubated overnight with primary antibodies of CRABP2 (Proteintech, Chicago, Illinois, USA) and β-Actin (Santa Cruz, Dallas, Texas, USA). Later, Immobilon Western chemiluminescent reagents (Millipore, billerica, MA, USA) were used to detect proteins on the membranes.
8
Western Blot Analysis of Autophagy Markers
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Whole cell lysates were prepared with RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (B14012, Bimake, USA). Protein concentrations were determined using the BCA protein assay (23229, Thermo Fisher Scientific, USA). Equal amounts of proteins were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to the appropriate PVDF membranes (0.22 µm: ISEQ00010; 0.45 µm: IPVH00010, Millipore, USA). The membranes were blocked with 5% nonfat dry milk at room temperature for 1 h and then incubated overnight at 4 °C with primary antibodies against Caveolin-1(16447-1-AP, Proteintech, USA), LC3 A/B (4108S, Cell Signaling Technology, USA), Beclin-1 (3495S, Cell Signaling Technology, USA), IRGM (AP11128b, Abgent, USA), p62 (sc-28359, Santa, USA) and β-actin (sc-58673, Santa, USA). Protein bands were visualized through Immobilon Western chemiluminescent reagents (WBKLS0500, Millipore, USA).
9
Immunoblot Analysis of Bcl2 in Glioma Cells
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Briefly, approximately 5 × 106 glioma cells were washed in PBS and lysed in lysis buffer (Thermo Scientific, Waltham, MA, USA). Then, lysates were incubated on ice for 30 min and centrifuged at 10,000×g for 30 min at 4 °C. Protein concentrations were calculated by a BCA kit (Thermo Scientific, Waltham, MA, USA). After sample separation by 15% SDS-PAGE, proteins were transferred onto NC membranes (HATF00010; Burlington, MA, Millipore). Membranes were incubated overnight at 4 °C for 1 h after blocking with primary antibodies as follows: Bcl2 (1:1,000, #2872; Cell Signaling Technology, Danvers, MA, USA) and tubulin (1:2,000; Proteintech, Chicago, IL, USA). Then, membranes were probed with secondary antibodies (1:10,000; Proteintech, Chicago, IL,USA). Bands were detected using Immobilon western chemiluminescent reagents (WBKLS0500; Millipore, Burlington, MA, USA).
10
Western Blot Analysis of SCAF11 Protein
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The cultured cells were collected and then lysed with IP lysis buffer supplemented with proteinase inhibitors. Equal amounts of total protein (30 μg) were loaded into each lane of 10% SDS–PAGE. Subsequently, proteins were transferred into PVDF membranes and blocked in 5% skimmed milk at RT for 2 h, and then incubated overnight at 4°C with primary antibodies in 5% BSA, followed by HRP-conjugated secondary antibody at RT for 1 h. Housekeep gene GAPDH served as an internal control. Primary antibodies included SCAF11 antibody (1:1,000, 28098-1-AP, proteintech) and GAPDH (1:10,000, 60004-1-Ig, proteintech). Finally, proteins were visualized using Immobilon Western chemiluminescent reagents (20020B6, Millipore, USA).
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