Total cell lysates from different experiments were obtained by lysing the cells in RIPA buffer (Wanlei Biotechnology, Beijing, China). The BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to quantify protein concentrations according to the manufacturer’s protocols. We used 10% SDS-PAGE to resolve 30 ug proteins and then transferred them to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies at 4°C for 12 h, followed by incubation with a horseradish peroxidase-conjugated goat-anti-rabbit or goat-anti-mouse secondary antibody (Cell Signaling Technology, CA, USA) at room temperature for 1 h. Specific bands were visualized using enhanced chemiluminescence detection (Thermo Fisher Scientific, Inc.). The signal intensity was determined with ImageJ software version 1.48. The antibodies used were: PIM1 (3247, Cell Signaling, 1: 2000), SND1 (ab65078, Abcam, 1: 1500), Phosphoserine (ab9332, Abcam, 1: 3000), and β-actin (PM053, MBL, 1: 5000). SA-β-gal activity was assessed as described previously [12 (link)].
Ab65078
Manufactured by Abcam
Sourced in United States, China
Ab65078 is a highly specific monoclonal antibody that recognizes the human interleukin-6 receptor (IL-6R). It is suitable for use in various research applications, including Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti α tubulin, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence detection, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab9332, by Abcam (1 mentions)
Ripa buffer, by Wanlei (1 mentions)
Ab189034, by Abcam (1 mentions)
Streptavidin peroxidase, by Zymo Research (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Rabbit anti α tubulin, by Abcam (1 mentions)
Alexa fluor 488 coupled antibodies against rabbit igg, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl system, by Yeasen (1 mentions)
Ab8227, by Abcam (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Odyssey clx imager, by LI COR (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
Blocking buffer, by LI COR (1 mentions)
8 protocols using ab65078
1
Western Blot and Senescence-associated β-Galactosidase Assay
2
IHC Analysis of SND1, PIM1, and CDKN2A
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Immunohistochemistry (IHC) was performed by the streptavidin-peroxidase method (Zymed Laboratories, Inc., San Francisco, CA, USA). The antibodies used were SND1 (ab65078, Abcam, 1: 50), PIM1 (ab200889, Abcam, 1: 50), and CDKN2A/p16INK4a (ab189034, Abcam, 1: 50). Immunostaining was carried out using a peroxidase-conjugated goat anti-rabbit secondary antibody according to the manufacturer’s instructions. Immunostaining was assessed and examined independently by 2 observers (LS Zhu and R Ding).
3
Immunofluorescence Assay of SND1 and Tubulin
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Immunofluorescence assays were conducted as previously described (Gao et al., 2010 ). Cells were untreated or treated with a 45°C heat shock for different times (0 min, 5 min, 15 min, 30 min, and 45 min). After fixation and permeabilization, cells were incubated with goat anti‐SND1 (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti‐α‐Tubulin (Abcam, Cambridge, UK) antibodies. Then, the Texas Red 596‐coupled anti‐goat polyclonal secondary antibodies to goat IgG H&L (Abcam) and Alexa Fluor 488‐coupled antibodies against rabbit IgG (Molecular Probes) were added for the fluorescence signal. The images and the fluorescence intensity profiles of the targeted regions were obtained with a Zeiss confocal microscope (Germany). At least 50 cells were examined.
In addition, for the Nocodazole treatment experiments, HeLa cells were pretreated with 2 μg mL−1 Nocodazole for 2 h and stimulated with or without the 45°C heat shock for 45 min. Immunofluorescence was performed using the rabbit anti‐SND1 (ab65078, Abcam) and mouse anti‐α‐Tubulin (T5168, Sigma) primary antibodies. After the fluorescent staining with Alexa Fluor 488‐coupled antibodies against rabbit IgG (Molecular Probes) and Alexa Fluor 546‐coupled anti‐mouse IgG antibody (Molecular Probes), an inverted Leica DMI6000 B microscope was used to collect the images. At least 50 cells were examined.
In addition, for the Nocodazole treatment experiments, HeLa cells were pretreated with 2 μg mL−1 Nocodazole for 2 h and stimulated with or without the 45°C heat shock for 45 min. Immunofluorescence was performed using the rabbit anti‐SND1 (ab65078, Abcam) and mouse anti‐α‐Tubulin (T5168, Sigma) primary antibodies. After the fluorescent staining with Alexa Fluor 488‐coupled antibodies against rabbit IgG (Molecular Probes) and Alexa Fluor 546‐coupled anti‐mouse IgG antibody (Molecular Probes), an inverted Leica DMI6000 B microscope was used to collect the images. At least 50 cells were examined.
4
LncRNA-HIT Interaction with p100 and CBP
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Approximately 1mg of E 11.5 limb bud lysates were used to immunoprecipitate p100 and CBP with endogenous LncRNA-HIT transcripts following the previously described RIP procedure [5 (link), 68 (link)] using antibodies specific for SND1 (p100) (Cat. Ab65078 Abcam, Cambridge, MA) and CBP (Cat. 7389 Cell Signaling, Danvers, MA). After immunoprecipitation of p100, CBP, or IgG, the co-immunoprecipitated RNA was extracted using TRIzol following the manufacturer’s protocol (ThermoFisher). After extraction, the isolated RNA was treated with RQ1 Rnase-Free Dnase (Cat. M6101 Promega, Madison, WI) and evaluated for enrichment of LncRNA-HIT using qRTPCR as described in the RNA isolation and qRTPCR section of the Methods. Three independent immunoprecipitation assays were performed for p100, CBP, and the IgG control analysis of LncRNA-HIT co-immunoprecipitation.
5
Western Blot for SND1 Protein Detection
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Cell lysates were resuspended in radio-immunoprecipitation assay dissolution buffer. The whole-cell lysates (30–60 μg in each well) were separated using 10% dodecylsulfate, sodium salt (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene fluoride membrane (Millipore, Danvers, MA, USA). The membrane was blocked with 5% skimmed milk and incubated overnight with primary antibodies against SND1 (ab65078) or β-actin (ab8227) at 4°C (Abcam, Cambridge, MA, USA). This was followed by the addition of a secondary antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The bands were observed by using an enhanced chemiluminescence (ECL) system (Yeasen, China).
6
Investigating Protein Interactions by Co-Immunoprecipitation
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To conduct co-immunoprecipitation experiments, 100 μg of either βTC3 or INS-1 832/13 nuclear extracts was combined with goat α-PDX1 antibody (Abcam, ab47383), α-SND1 antibody (PA5–40124), and either goat or rabbit IgG. The mixture was then incubated overnight at 4°C. The following day, Protein A/G PLUS-agarose was used to precipitate the antibody complexes, which were subsequently washed with PBS and boiled with SDS loading dye. The immunoprecipitants were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with Blocking Buffer (LI-COR, 927–70001). Membranes were probed with primary antibodies: goat α-Pdx1 (Abcam, ab47383; 1:20000) and rabbit α-SND1 (Abcam, ab65078; 1:500). For detection, LI-COR IRDye secondary antibodies were employed, and fluorometric scanning was carried out using an Odyssey CLx Imager. Finally, ImageJ software was utilized to quantify the band intensity.
7
Investigating Cellular Stress Responses
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Western blot analysis was conducted as previously described (Gao et al., 2010 ; Su et al., 2015 ). Briefly, HeLa cells were treated or untreated at 45°C for 45 min after the pretreatment with or without 2 μg mL−1 Nocodazole for 2 h. The total cell lysates were harvested and subjected to SDS‐PAGE. The following antibodies were used: rabbit anti‐SND1 (ab65078, Abcam), mouse anti‐α‐Tubulin (T5168, Sigma), mouse monoclonal anti‐β‐actin antibody (A1978, Sigma Aldrich), rabbit anti‐eIF2α (#5324; Cell Signaling Technology, Beverly, MA) and rabbit anti‐p‐eIF2α (Ser 51) (#3597; Cell Signaling Technology). The ImageJ 2X software (NIMH, Bethesda, MD) was used to measure the grayscale value of the band. The level of p‐eIF2α (Ser 51) was normalized to the total eIF2α protein.
8
Immunohistochemical Analysis of SND1 in OS
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IHC of OS tissues and adjacent normal tissues was performed. Tissues were fixed using formalin, embedded in paraffin, and then serially sectioned at 4 μm. Slides were then incubated with anti-SND1 antibody (ab65078, Abcam, Shanghai, China) overnight in a moist chamber at 4°C, washed in phosphate buffer saline (PBS), and then incubated with corresponding secondary antibodies. Slides were developed with diaminobenzidine and counterstained with hematoxylin.
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