Anti lamin b sc 6216

Protein extracts were subjected to SDS-PAGE (8–12% gels) and blotted onto PVDF membranes. After blocking with 5% fat-free milk, the membranes were incubated with the following antibodies at 4 °C overnight: anti-OPTN (sc-166576, 1:1000), anti-TLR4 (sc-293072, 1:1000), and anti-Lamin B (sc-6216, 1:1000) were purchased from Santa Cruz; anti-JAK2 (#3230, 1:1000), anti-p-JAK2(Tyr1007/1008) (#3771, 1:1000), anti-STAT3 (#9139, 1:1000), anti-p-STAT3(Y705) (#9145, 1:1000), anti-MyD88 (#4283, 1:1000), anti-p-NF-κB (#3033, 1:1000), anti-HA (#3724, 1:1000), and anti-Flag (#86861, 1:1000) were purchased from Cell Signaling Technology; anti-NF-κB (ET1603-12, 1:1000), anti-p38 MAPK (ET1602-26, 1:1000), anti-p-p38 MAPK (ER1903-01, 1:1000), anti-IRF3 (ET1612-14, 1:1000), and anti-p-IRF3(S386) (ET1608-22, 1:1000) were purchased from Huabio; anti-GAPDH (db106, 1:5000) was purchased from DiagBio Technology. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated IgG (MULTI Sciences) and visualized with enhanced chemiluminescence (ECL, PerkinElmer) detection reagents (Thermo Scientific, USA). GAPDH or Lamin B was used as a loading control. Images were taken by GE AI600.

The following antibodies and reagents were used: anti-LAMP1 (ab24170, abcam), anti-LAMP2 (clone H4B4, Developmental Studies Hybridoma Bank), anti-Lamin B (sc-6216, Santa Cruz), anti-Rab11 (clone 47, BD Biosciences), anti-GAPDH (6C5, EMD Millipore), anti-DYKDDDDK (clone L5, Novus Biologicals), anti-myc (4A6, Merck Millipore) (562, MBL Life Science), anti-TGN46 (AHP500GT, Bio-Rad), anti-integrin beta 1 (ab183666, abcam), anti-EEA1 (610456, BD Biosciences), anti-Transferrin receptor (3C11F11, proteintech), anti-mouse IgG HRP linked whole antibody from sheep (NA931V, GE Healthcare), anti-rabbit IgG HRP linked whole antibody from donkey (NA934V, GE Healthcare), anti-rat IgG HRP linked whole antibody from goat (NA935, GE Healthcare), Hoechst 33342 (Dojindo), donkey anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 594 (A-21203, Invitrogen), donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (A-11008, Invitrogen), donkey anti-rat IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 (A-21208, Invitrogen), Donkey anti-Sheep IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A-11016, Invitrogen). Biotinylated PhoSL was kindly provided by J Chemical (58 (link)), and other biotinylated lectins were purchased from J Chemical (ConA, #J203; L4-PHA, #J212; SSA, #J218; MAM, #J210).

Protein samples (20–40 g) were loaded on SDS-PAGE and transferred to nitrocellulose filters. After blocking the nonspecific background in 5% non-fat milk, nitrocellulose membranes were incubated at +4 °C overnight with anti-GATA4 (sc-1237) or anti-Lamin B (sc-6216) (Santa Cruz Biotechnology). To detect phosphorylated GATA4 the membrane was blocked in 5% non-fat milk overnight + 4 °C and incubated with anti-phospho-GATA4 (Ser105) (44-948, Invitrogen) for 2 h at room temperature. After washing, the filters were incubated for 1 hour with an HRP-conjugated anti-rabbit, anti-mouse or anti-goat secondary antibody. The protein amounts were detected by enhanced chemiluminescence with ECL Plus reagents (RPN2132, Amersham Biosciences) followed by digitalization of chemiluminescence with Luminescent Imager Analyzer LAS-3000 (Fujifilm) and analyzing with Quantity One software (Bio-Rad Laboratories). For infrared detection, the secondary fluorescent antibodies were Alexa Fluor 680 from Invitrogen (Life Technologies Ltd, Paisley, UK) and IRDye 800 from Rockland Immunochemicals (Gilbertsville, PA, USA). Antibody binding was detected by the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). For a second immunoblotting, the membrane was stripped for 30 min at + 60 °C in stripping buffer (62.5 mM Tris pH 6.8, 2% SDS and 100 mM β -mercaptoethanol).

Western blot analysis was conducted on the basis of a previously described method [64 (link)]. Protein extracts of mammospheres derived from MDA-MB-231 cells (1 × 10⁴ per well) treated with dihydroconiferyl ferulate (50 μM) were analyzed using SDS-PAGE (8%) and electrotransferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated at RT for 1 h in Odyssey blocking buffer (LI-COR, Lincoln, NB, USA) containing Tween 20 (0.1%, v/v). The membranes were then incubated overnight at 4 °C in a blocking buffer containing the following primary antibodies: anti-EGFR (#4267s, Cell Signaling Technology, Denver, CO, USA), anti-pStat3 (#9145s, Cell Signaling Technology, Denver, CO, USA), anti-c-Myc (#5605s, Cell Signaling Technology, Denver, CO, USA), anti-Stat3 (sc-482), anti-Lamin B (sc-6216), and anti-β-actin (sc-47778 Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were washed with PBST (phosphate-buffered saline with Tween 20, 0.1%, v/v) and incubated with anti-rabbit (IRDye 800CW-conjugated), anti-goat (IRDye 680RD-conjugated), or anti-mouse (IRDye 680RD-conjugated) secondary antibodies. An ODYSSEY CLx machine (LI-COR, Lincoln, NB, USA) was used to examine the band signals.

Primary antibodies used for immunoelectron microscopy, chromatin immunoprecipitation and immunofluorescence included, anti-trimethyl-HistoneH3Lys9 (07–442), anti-acetyl-HistoneH3Lys9 (06–942) and anti-γH2AX (05–636) from Upstate; anti-Tau1 (MAB3420), anti-NeuN (MAB377) and anti-phospho-Tau AT8 (MN1020) from Pierce; anti-H3 (ab24834) from Abcam; anti Tau5 (AHB0042) from Biosource; anti-HP1α (2HP-1H5-AS) from Euromedex; anti-lamin B (sc-6216) from Santa Cruz; mouse anti-NSs polyclonal antibodies48 (link). Primary antibodies used for western blot included, anti-trimethyl-HistoneH3Lys9 (07–442) and anti-γH2AX (05–636) from Upstate; anti β-actin from Sigma, anti-H3 (ab24834) from Abcam; anti-HP1α (2HP-2G9-AS) from Euromedex. The secondary antibodies used for immunofluorescence and immuno-FISH were the Alexa 488-conjugated chicken anti-mouse (A21200), Alexa 555-conjugated donkey anti-mouse (A31570), and Alexa 555-conjugated donkey anti-rabbit (A31572) antibodies from Molecular Probes. The secondary antibodies used for western blot were the peroxydase horse anti-mouse (PI-2000) and peroxydase goat anti-rabbit (PI-1000) IgG antibodies from Vector laboratories.

The anti‐PELI1 (sc‐271065), anti‐NFκB p100/p52 (sc‐7386), anti‐RelB (sc‐48366), and anti‐Lamin B (sc‐6216) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and anti‐NIK (4994), anti‐ACTIN (4970), anti‐Cleaved PARP (5625), anti‐Cleaved Caspase‐3 (9664), anti‐Cleaved Caspase‐7 (8438), anti‐Cleaved Caspase‐9 (52873), anti‐Bcl‐XL (2764), anti‐Bax (2772), anti‐Bcl‐2 (15071), anti‐Bak (12105), anti‐Mcl‐1(5453), anti‐A1/Bfl‐1 (D1A1C) (14093), anti‐Bcl‐w (31H4) (2724), anti‐Phospho‐NF‐κB p65 (Ser536) (3033), and anti‐Phospho‐Akt (Ser473) (4060) anti‐p53 (2527), anti‐Phospho‐p53 (Ser15) (9284), anti‐ATM (2873), anti‐Phospho‐ATM (Ser1981) (13050), anti‐NF‐κB1 p105 (4717), anti‐c‐Rel (4727), anti‐Phospho‐NF‐κB2 p100/p52 (Ser866/870) (4810), and anti‐Phospho‐RelB (Ser552) (D41B9) (5025) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti‐Lys48 ubiquitin (051307) antibody was purchased from Millipore (Burlington, MA, USA). MG132 (C2211) and 4‐NQO (N8141) were purchased from Sigma (St. Louis, MO, USA). Puromycin was purchased from Merck (54041, Kenilworth, NJ, USA). Cycloheximide (HY‐12320) was purchased from MCE. ¹⁸F‐FDG and ¹⁸F‐ML‐10 were synthesized by Jiangsu Huayi Technology Co., Ltd (Suzhou, China). The radiochemical purity was more than 95%.