MG-63 and Saos-2 cells infected with LV-shCNN3 or LV-NC were seeded (4 × 105 cells/well) in 6-well plates. After being cultured for 48 h, cells were harvested, and the total protein content was isolated with RIPA buffer. After protein quantification, 30 μg of total protein was used for western blotting, which was conducted according to the methods described previously [33 (link)]. The primary antibodies used in this study were purchased from Abcam (Cambridge, MA, USA) and their details are as follows: anti-CNN3 (1:2000, ab151427), anti-MMP9 (1:1500, ab76003), anti-VEGF (1:5000, ab52917), anti-vimentin (1:1000, ab92547), anti-E-cadherin (1:1000, ab231303), p-p38 (1:1000, ab45381), p38 (1:1000, ab32142), ERK1/2 (1:1000, ab115799), p-ERK1/2 (1:800, ab214362), and anti-GAPDH (1:5000, ab181602).
Ab32142
Manufactured by Abcam
Sourced in United States
Ab32142 is a lab equipment product from Abcam. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab214362, by Abcam (1 mentions)
Ab115799, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab231303, by Abcam (1 mentions)
Ab45381, by Abcam (1 mentions)
Ab52917, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab68477, by Abcam (1 mentions)
Ab62352, by Abcam (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Ab208035, by Abcam (1 mentions)
Ab178867, by Abcam (1 mentions)
Enhanced chemiluminescence system, by Beyotime (1 mentions)
Ab17942, by Abcam (1 mentions)
Ab18197, by Abcam (1 mentions)
Ab223500, by Abcam (1 mentions)
Ab76572, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab182733, by Abcam (1 mentions)
5 protocols using ab32142
1
Western Blot Analysis of CNN3 Regulation
2
Protein expression analysis using Western blot
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The brain tissues were lysed using RIPA buffer (Cat#R0010, Solarbio, China) and sonicated to obtain proteins solution. Proteins concentration was determined by the Bradford method. 10% SDS-PAGE gel was used to separate proteins, which was then transferred to the PVDF membrane (CAT. NO. ISEQ00010). The membranes were blocked with 5% skimmed milk in TBS tween-20 (TBST), then incubated with the primary antibodies [Nrf2 (ab62352, abcam), P38MAPK (ab32142, abcam) heme oxygenase-1 (HO-1) (ab68477, abcam)], diluted in TBST at 4°C overnight, and then incubated with the HRP-conjugated secondary antibody at room temperature for 2 h. The images were detected using Bio-Rad ChemiDoc XTS+ system [28 (link)].
3
Protein Extraction and Western Blot Analysis
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Tissues or cells were lysed in RIPA lysis buffer (Beyotime) to extract total proteins. After separation by 12% SDS‐PAGE, proteins were transferred onto PVDF membranes (Bio‐Rad) and then blocked in 5% skim milk. The membranes were next incubated with the primary and secondary antibodies, including anti‐MAP4K3 (#92427; Cell Signaling Technology), antiproliferating cell nuclear antigen (anti‐PCNA; ab18197; Abcam), anti‐Bcl‐2‐associated X protein (anti‐Bax; ab182733), anti‐ERK1/2 (ab17942; Abcam), anti‐p‐ERK1/2 (ab223500; Abcam), anti‐JNK (AB208035; Abcam), anti‐p‐JNK (ab76572; Abcam), anti‐p38 (ab32142; Abcam), anti‐p‐p38 (ab178867; Abcam), anti‐GAPDH (ab9485; Abcam) and goat‐anti rabbit (ab205718; Abcam). The protein signals were detected using an enhanced chemiluminescence system (Beyotime).
4
Western Blot Analysis of Cell Signaling Proteins
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Whole-cell extracts were prepared in lysis buffer and the concentration of proteins was determined using the BCA Protein Assay Reagent Kit (Thermo scientific). Proteins were separated in 10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked using TBST buffer containing 5% (w/v) non-fat milk 1 h at RT and probed with specific antibodies (1 : 1000) for CDC7 (sc-56275, Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-3 (active) (1476-1; Epitomics, CA, USA), p38 (Y122) (ab32142; Abcam), phospho-p38 MAPK (pT180/pY182) (1229S; Epitomics), MCM2 (CST-3619; Cell Signaling), phospho-MCM2 (Ser139) (CST-12958), PARP3(ab96601), DDIT4(ab63059), ATM (CST-2873S), ATM phospho (pS1981) (2152-1), ATR (N19) (sc-1887), phospho-ATR (Ser428) (CST-2853S), cyclin D1 (ab24249), p53 (CST-2527), phospho-p53 (Ser15) (CST-9284), phospho-p53 (Ser20) (CST-9287), phospho-p53 (Ser46) (CST-2521), Ac-p53 (Lys382) (CST-2525), β-actin (PM053; MBL, Nagoya, Japan) and α-tubulin (PM054; MBL). Blots were developed with a secondary antibody (IgG)-conjugated horseradish peroxidase. Chemiluminescence signals were visualized using SuperSignal1 West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).
5
Signaling Pathway Inhibition Assay
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Peprotech (Rocky Hill, NJ, USA) supplied us with recombinant rat IL-1β. PKC-δ inhibitor rottlerin was obtained from MedChemExpress (Monmouth Junction, NJ). Abcam (Cambridge, MA, USA) offers PKC-δ (ab182126), JNK (ab124956), and p38 (ab32142) antibodies. The Cell Signaling Technology (Beverly, MA, USA) provided antibodies against p-PKC-δ (2055S) and p-p38 (4511S). Proteintech (ProteinTech Group, Chicago, IL, USA) provided antibodies against p-JNK (80024-1-RR), BAX(50599-2-Ig), caspase-3(66470-2-IG), and GAPDH (10494-1-AP).
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